Type VIII collagen is a matrix protein expressed in a number of tissues undergoing active remodeling, including injured arteries during neointimal formation and in human atherosclerotic plaques; however, very little is known about its function. We have investigated whether the type VIII collagen stimulates smooth muscle cell (SMC) migration and invasion by binding to integrin receptors and up-regulating matrix metalloproteinase (MMP) production. SMCs attached to plates coated with type VIII collagen in a dose-dependent manner, with maximal attachment occurring with coating solutions containing 25 microgram/ml collagen. Type VIII collagen at 100 microgram/ml stimulated an 83-fold increase in the migration of SMCs in a chemotaxis chamber. Antibodies against beta1 integrin receptors prevented attachment and migration of SMCs. Antibodies against alpha1 or alpha2 integrins reduced attachment of SMCs to type VIII collagen by 29% and 77%, respectively. We found that SMCs grown from the rat neointima, but not medial SMCs, increased their production of MMP-2 and -9 on adherence to type VIII collagen. This suggests that there is an important difference in phenotype between intimal and medial SMCs and that intimal SMCs have distinct matrix-dependent signaling mechanisms. Our findings suggest that type VIII collagen deposited in vascular lesions functions to promote SMC attachment and chemotaxis, and signals through integrin receptors to stimulate MMP synthesis, all of which are important mechanisms used in cell migration and invasion.

Remodeling of injured blood vessels is dependent on smooth muscle cells and matrix metalloproteinase activity. Doxycycline is a broad spectrum matrix metalloproteinase inhibitor that is under investigation for the treatment of acute coronary syndromes and aneurysms. In the present study, we examine the mechanisms by which doxycycline inhibits smooth muscle cell responses using a series of in vitro assays that mimic critical steps in pathological vascular remodeling. Doxycycline treatment dramatically increased smooth muscle cell adhesion to the substrate, as evidenced by interference reflection microscopy and immunostaining for paxillin and phosphotyrosine. Cell aggregation was also potentiated after treatment with doxycycline. Treatment with 104 mumol/L doxycycline reduced thymidine uptake by 58% compared with untreated cells (P < 0.05) and inhibited closure of a scrape wound made in a smooth muscle cell monolayer by 20% (P < 0.05). Contraction of a three-dimensional collagen gel was used as an in vitro model for constrictive vessel remodeling, demonstrating that treatment with 416 mumol/L doxycycline for 12 hours inhibited collagen gel remodeling by 37% relative to control (P < 0.05). In conclusion, we have shown that doxycycline treatment leads to dramatically increased smooth muscle cell adhesion, which in turn might limit responses in pathological vascular remodeling.

Abstract-This study tests the hypothesis that ␣ v ␤ 3 integrin receptors play a critical role in smooth muscle cell (SMC) migration after arterial injury and facilitate migration through the upregulation of matrix metalloproteinase (MMP) activity. We showed that ␤ 3 integrin mRNA was upregulated by SMCs in the balloon-injured rat carotid artery in coincidence with MMP-1 expression and early SMC migration. Treatment with the ␤ 3 integrin-blocking antibody F11 significantly decreased SMC migration into the intima at 4 days after injury, from 110.8Ϯ30.8 cells/mm 2 in control rats to 10.29Ϯ7.03 cells/mm 2 in F11-treated rats (Pϭ0.008). By contrast, there was no effect on medial SMC proliferation or on medial SMC number in the carotid artery at 4 days. In vitro, we found that human newborn SMCs produced MMP-1 but that adult SMCs did not. This was possibly due to the fact that newborn SMCs expressed ␣ v ␤ 3 integrin receptors, whereas adult SMCs did not. Stimulation of newborn (␣ v ␤ 3 ϩ) SMCs with osteopontin, a matrix ligand for ␣ v ␤ 3 , increased MMP-1 production from 114.4Ϯ35.8 ng/mL at 0 nmol/L osteopontin to 232.5Ϯ57.5 ng/mL at 100 nmol/L osteopontin. Finally, we showed that stimulation of newborn SMCs with platelet-derived growth factor-BB and osteopontin together increased the SMC production of MMP-9. Thus, our results support the hypothesis that SMC ␣ v ␤ 3 integrin receptors play an important role in regulating migration by stimulating SMC MMP production. We and other investigators have shown that matrix metalloproteinase (MMP) activity is necessary for SMC migration; MMP-2, MMP-3, MMP-9, and membrane-type MMP-1 (MT-MMP-1) are upregulated coincident with SMC migration during the first week after balloon injury in several species, 12 and treatment with specific MMP inhibitors dramatically attenuates SMC migration in vivo. 13,14 Studies with fibroblasts, keratinocytes, and melanoma cells have demonstrated that MMP production is regulated by feedback from the extracellular matrix through integrin receptor signaling. 15 We have investigated the possibility that SMCs are susceptible to similar feedback from the matrix, particularly by osteopontin, a ligand for the ␣ v ␤ 3 receptor.We demonstrated that ␤ 3 integrin mRNA was upregulated after balloon catheter injury of the rat carotid artery and that MMP-1 protein in the vessel wall was increased in parallel with ␤ 3 integrin expression. Treatment with the ␤ 3 integrin-blocking antibody (F11) 16 caused a reduction in SMC migration, but not proliferation, 4 days after balloon catheter injury. Finally, we showed that osteopontin alone and in coordination with platelet-derived growth factor (PDGF)-BB stimulated MMP activity in SMCs, and we also demonstrated the critical importance of the ␣ v ␤ 3 integrin receptor in mediating this response. MethodsAll chemicals were obtained from Sigma Chemical Co unless stated otherwise. Surgery and Antibody TreatmentIn vitro adhesion assays were performed to determine whether F11 blocked ␣ v ␤ 3 in rat carotid SMCs. Tissue cultur...

Objective-Type VIII collagen is upregulated after vascular injury and in atherosclerosis. However, the role of type VIII collagen endogenously expressed by smooth muscle cells (SMCs) and in the context of the vascular matrix microenvironment, which is rich in type I collagen, is not known. To address this, we have compared aortic SMCs from wild-type (WT) mice to SMCs from type VIII collagen-deficient (KO) mice when plated on type I collagen. Methods and Results-Type VIII collagen was upregulated after wounding of WT SMCs. KO SMCs exhibited greater adhesion to type I collagen than WT SMCs (optical density [OD 595 ]ϭ0.458Ϯ0.044 versus 0.193Ϯ0.071). By contrast, the WT SMCs spread more (389Ϯ75% versus 108Ϯ14% increase in cell area), migrated further (total distance 80.6Ϯ6.2 m versus 64.2Ϯ4.4 m), and exhibited increased [ 3 H]-thymidine uptake (160 000Ϯ22 300 versus 63 100Ϯ12 100 counts per minute) when compared with KO SMCs. Gelatin zymograms showed that WT SMCs expressed latent matrix metalloproteinase 2, whereas KO SMCs did not. Addition of exogenous type VIII collagen returned levels of KO SMC adhesion (OD 595 ϭ0.316Ϯ0.038), migration (79.5Ϯ5.8 m), and latent matrix metalloproteinase 2 expression to levels comparable to WT SMCs. Conclusions-This study suggests that SMCs can modify the matrix microenvironment by producing type VIII collagen, using it to overlay type I collagen, and generating a substrate favorable for migration. (Arterioscler Thromb Vasc Biol. 2006;26:56-61.)Key Words: atherosclerosis Ⅲ restenosis Ⅲ collagen Ⅲ smooth muscle cell migration Ⅲ MMP C ollagens compose a large portion of the extracellular matrix in the vessel wall and play important roles maintaining the strength and structural integrity of blood vessels. During atherosclerosis, collagen accumulation contributes to lesion growth and vessel contraction, and collagen degradation leads to plaque instability and rupture. 1 In addition to their roles providing structural support in the arterial wall, in vitro studies suggest that collagens can act as signaling molecules stimulating changes in the phenotype and behavior of smooth muscle cells (SMCs), endothelial cells, and macrophages. Recent research suggests that SMCs respond differently to different types or physical states of collagen. For example, intact type I collagen maintains SMC quiescence, whereas denatured or degraded collagen stimulates proliferation and migration. [2][3][4] Type VIII collagen is a member of the short chain collagen family, composed of ␣1 (VIII) and ␣2 (VIII) chains. It is produced by corneal and aortic endothelial cells, as well as mesangial cells, and is upregulated during the proliferation of these cells and during angiogenesis. [5][6][7][8] In normal arteries, it has been localized to the subendothelial intima and is present in very low amounts in the media and adventitia. 9 Type VIII collagen expression is dramatically increased after balloon injury of the rat carotid 10,11 or the porcine coronary artery. 12 In the rat balloon injury model, type VIII coll...

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