In the present study, we tested the hypothesis that OxPLs modulate ECM gene expression in VSMCs. Consistent with this hypothesis, results of real-time RT-PCR analyses of the candidate genes and gene microarray studies showed that POVPC altered expression of a number of ECM or ECM-related genes, including multiple collagens, fibronectin 3, laminin μ2, integrins, and the proteoglycan versican. We also demonstrated that OxPLs, and in particular POVPC, markedly upregulated type VIII collagen expression in cultured VSMCs, as well as in vivo within carotid arteries.
Previous studies have shown that type VIII collagen expression is dramatically increased within VSMCs in response to vascular injury,24,25
as well as in atherosclerotic plaques of the apoE
Type VIII collagen has been shown to stimulate attachment, focal adhesion formation, and chemotaxis of cultured SMC, as well as to modify matrix metalloproteinase synthesis.23
Furthermore, it has been previously shown that VSMCs from type VIII collagen–deficient mice exhibit greater adhesion to type I collagen than WT VSMCs. In contrast, WT VSMCs spread more, migrate further, exhibit increased proliferation, and express higher level of matrix metalloproteinase 2 in comparison with type VIII collagen–deficient VSMCs,22
suggesting that the de novo production of type VIII collagen allows VCMCs to overcome adhesion to type I collagen. A number of factors have been shown to modulate type VIII collagen expression in cultured VSMCs including platelet-derived growth factor-BB,24,25
basic fibroblast growth factor-2,26
transforming growth factor β
and colony-stimulating factor,27
which increase expression, and interferon γ
which decreases expression. However, virtually nothing is known regarding factors that regulate the expression of type VIII collagen or other ECM proteins by VSMCs within atherosclerotic lesions.
Of particular relevance to the present studies, previous studies by Plenz et al28
showed reduced type VIII collagen expression in the media and adventitia, but increased expression in the intima, of carotid arteries in rabbits fed a high-cholesterol diet. However, no direct evidence was presented that these effects were mediated by OxPLs. Our present results clearly demonstrate that OxPAPC, as well as its component phospholipids PGPC, PEIPC, and POVPC, significantly increase type VIII collagen in vitro. Of major significance, we also showed that POVPC increased type VIII collagen expression within carotid arteries in vivo. Taken together, results suggest that the oxidized arachidonoyl phospholipids may be somewhat unique among modified lipids in inducing expression of type VIII collagen and thus implicate these factors as potential mediators of increased type VIII collagen expression in atherosclerotic lesions. A key unresolved question is whether POVPC or other OxPLs directly mediate SMC phenotypic switching in vivo including inducing alterations in expression of type VIII collagen and other ECM components. However, such experiments are not feasible at present for several reasons including: (1) OxPL receptors have not been identified for precluding use of either pharmacological or genetic loss-of-function approaches; (2) there is a lack of highly specific and efficacious inhibitors to block formation of specific OxPL species; and (3) although there are a number of neutralizing antibodies to specific OxPL species,29
it is not clear these can maintain efficacious inhibition in vascular lesions over long periods of time in vivo, and, of course, such approaches would not be selective in inhibiting OxPL responses within VSMCs.
Results of the present study also provide novel insights regarding mechanisms that regulate type VIII collagen expression, in that we show that effects of POVPC are dependent on Sp1-induced activation of Klf4. Klf4 is a member of the Krüppel family of transcription factors.30
Studies in our laboratory have shown that Klf4
expression is normally undetectable in SMC but is rapidly increased in vivo following vascular injury.31,32
Moreover, it was demonstrated that the platelet-derived growth factor-BB– and POVPC-induced downregulation of SMC marker genes is mediated, in part, by Klf4.7,31
In addition, our present results demonstrated that Klf4
expression are increased in the aortas of apoE
KO mice after 13 weeks of Western diet feeding. Finally, results of recent studies in our laboratory showed that conditional knockout of Klf4
in adult mice resulted in a transient delay in downregulation of SMC marker genes, but subsequently enhanced neointimal formation following ligation induced injury of the carotid artery.32
That is, Klf4 appears to be a key rate-limiting factor for initial phenotypic switching of SMC in response to vascular injury but also plays a key role in negative regulation of SMC growth. We found that Klf4 is required for POVPC-induced Col8a1
expression in vivo and in vitro, as well as LAMA2
expression in vitro. Based on the results of the present studies, it is interesting to speculate that Klf4 might also be a key regulator of ECM expression in VSMCs during atherogenesis.
Of interest, although we demonstrated that Sp1 was required for POVPC-induced increases in Klf4
expression, we did not find a significant increase in Sp1 expression or synthesis after POVPC treatment (OA Cherepanova, GK Owens, unpublished data, 2008), suggesting that effects might involve posttranscriptional modification of Sp1 and/or its ability to bind to the Klf4
promoter. Consistent with this possibility, Sp1 has been shown to undergo various posttranscriptional modifications including phosphorylation and O
-glycosylation (reviewed by Black et al33
) that increase its transcriptional activity, at least in part, by enhancing its binding to DNA.
Previously, we reported that POVPC treatment of cultured rat aortic SMC resulted in enhanced repair in a scratch wound assay.7
Results of the present studies showed that POVPC and PEIPC, but not PGPC, enhanced VSMC migration. Consistent with these observations, previous studies have shown differential effects of POVPC versus PGPC on monocyte and neutrophil binding to endothelium.15,34
Moreover, recent voltage-clamp studies by Leitinger et al34
suggested that POVPC and PGPC recognized different receptors based on mRNA expression studies in Xenopus
oocytes. Taken together, results suggest that the stoichiometry of specific OxPLs within lesions may have important functional consequences through differential effects on VSMCs, as well as other vascular cells.
Previous studies by Rocnik et al35
suggested that new collagen synthesis is required for VSMC migration on type I collagen–covered substrata. Results of the present studies showing that POVPC-induced migration of cultured rat aortic SMC was inhibited by siRNA suppression of type VIII collagen and failed to occur in type VIII KO mouse aortic SMC provide evidence that de novo synthesis of type VIII collagen plays an important role in mediating VSMC migration in vitro. Moreover, we also showed that POVPC-induced VSMC migration was dependent on Klf4. These findings are highly intriguing in that they raise the question as to whether Klf4-dependent SMC migration is mediated exclusively through induction of type VIII collagen or whether Klf4 also activates additional signaling pathways important in this response.
Interestingly, we found that POVPC did not induce and actually inhibited VSMC migration at concentrations of >5 μ
g/mL. From our recent studies,7
we know that this concentration is not toxic for VSMCs. As such, it is possible that activation of additional molecular pathways might be responsible for the inhibition of VSMC migration by the higher concentrations of POVPC.
In conclusion, we provide novel evidence showing that OxPLs activate coordinate expression of a variety of ECM genes in VSMCs including type VIII collagen and that activation of type VIII collagen is dependent on both Klf4 and Sp1. Moreover, we show that increased type VIII collagen expression is required for POVPC-induced migration of VSMCs, indicating that activation of ECM genes has important functional consequences. Future studies are needed to define specific receptors and downstream effector molecules that mediate the effects of specific OxPLs in vivo.