Kruppel-like Factor 15 Regulates Smooth Muscle Response to Vascular Injury—Brief Report
To determine the role of Kruppel-like factor (KLF) 15, a zinc finger transcriptional factor that is expressed in vascular smooth muscle cells (VSMCs) in vascular biology. VSMCs respond to mechanical injury via a tightly orchestrated series of gene regulatory events. KLF15 is broadly expressed in both arterial and venous vascular beds in a VSMC restricted fashion. KLF15 expression is markedly reduced by both pharmacological and mechanical stimuli. To examine the specific role of KLF15 in the vascular response to injury, we performed femoral artery wire injury in KLF15−/− and wild-type mice. KLF15−/− mice develop exaggerated neointimal growth, with evidence of increased SMC proliferation and migration within the neointima. In concordance, gain and loss of function studies in isolated VSMCs demonstrate that KLF15 can directly inhibit SMC proliferation and migration. To our knowledge, these data are the first to identify KLF15 as a novel inhibitor of VSMC proliferation and migration and to implicate this factor as a critical regulator of the vascular response to injury.
In response to injury, vascular smooth muscle cells (VSMCs) undergo marked phenotypic changes, characterized by enhanced cellular proliferation, migration, and extracellular response, that contribute to disease processes such as restenosis or transplantation arteriopathy. The transcriptional events leading to the highly orchestrated process of VSMC activation, proliferation, migration, and neointimal formation have been the subject of intense study; however, they remain incompletely defined.1–4
Kruppel-like factors (KLFs) are a subclass of the zinc finger family of transcriptional regulators that are key regulators of gene expression, cell growth, and differentiation.5,6 Our laboratory identified KLF15 as a novel member of this family that plays critical roles in cardiomyocyte, adipocyte, and hepatocyte biology.6,7 However, its role in VSMCs has yet to be determined. In the current study, we report that KLF15 is robustly expressed in VSMCs across arterial and venous beds. Furthermore, our in vitro and in vivo observations identify KLF15 as a novel and essential regulator of VSMC response to injury.
The KLF15−/− mouse was generated as previously described.7 Mouse femoral artery wire injury was performed in wild-type and KLF15−/− mice (n=17 in each group), and histopathologic analyses were performed after 2 weeks. RNA isolation, RT-PCR, and Northern blotting were performed as previously described.7 VSMC migration was examined by the Boyden chamber assay, and DNA synthetic rate was determined by the [3H]thymidine incorporation assay. Data are expressed as mean±SD. Differences between experimental groups were evaluated for statistical significance by using the t test for unpaired data. P<0.05 was considered statistically significant.
KLF15 Regulates the Biological Response to Vascular Injury
By using a KLF15−/+ reporter mouse in which the mouse KLF15 locus was targeted using a nuclear-localized B-galactosidase cassette,7 we examined vascular KLF15 expression using in situ 5-bromo-4-chloro-3-indolyl β-d-galactosidase staining. KLF15 is abundantly expressed in both the arterial and venous VSMC layer (Figure 1A). To gain initial insights into KLF15 function, we assessed its expression after pathological stimuli in vitro and in vivo. KLF15 mRNA expression was markedly reduced in cultured rat aortic SMCs in response to platelet-derived growth factor-BB (PDGF-BB), a potent inducer of SMC proliferation and migration (Figure 1B, left panel, representative Northern blot of 4 independent experiments; Figure 1B, right panel, RT-PCR of KLF15/18S ribosomal RNA; n=3 in each group). Similarly, we observed a marked reduction in KLF15 mRNA at 5 days in wire-injured femoral arteries (approximately 80%) (n=7 in the sham group and n=5 in the injured group) (Figure 1C).
To determine whether KLF15 directly modulates the biological response to vascular injury, we performed femoral artery wire injury in KLF15−/− mice and wild-type littermate controls. Fourteen days after wire injury, KLF15−/− mice developed enhanced intimal growth, with a 170% increase (P=0.02) in intimal area (Figure 1D). The intimal to medial area ratio was increased significantly in KLF15−/− compared with wild-type mice ([data given as mean±SD] 0.98±0.47 versus 0.65±0.41; P=0.01). At 14 days, SMC α-actin and leukocyte CD45 staining confirmed that the dominant cellular contributor to the neointima was VSMCs (Figure 1D and data not shown for CD45 staining). Intimal cellular proliferation was assessed by incorporation of 5-bromodeoxyuridine (BrdU) and was increased by 167% (P=0.045) in KLF15−/− mice (Figure 1D). Quantitative morphometric data are summarized in the supplemental Table (available online at http://atvb.ahajournals.org).
KLF15 Regulates VSMC Proliferation and Migration
Because KLF15−/− femoral arteries demonstrated excess neointimal growth (Figure 1D), we hypothesized that KLF15 might directly regulate VSMC proliferation and migration. KLF15−/− mouse aortic SMCs exhibited significantly increased proliferation and migration, both at baseline and in response to PDGF-BB stimulation (Figure 2A and C). Conversely, adenoviral overexpression of KLF15 in rat aortic SMCs strongly inhibited PDGF-BB–induced cellular proliferation and migration (Figure 2B and D).
To our knowledge, the data presented herein are the first to identify KLF15 as an essential regulator of the biological response to vascular injury. Our studies indicate that KLF15 is a novel inhibitor of SMC migration and proliferation. Consistent with these observations, KLF15−/− mice exhibited enhanced neointimal formation after wire injury. Our data also indicate that the effects observed are likely the result of a cell-intrinsic defect, as evidenced by the fact that KLF15−/− VSMCs exhibit augmented proliferation and migration. Taken together, these studies suggest that KLF15 positively regulates the vascular response to injury.
Our observations add to an increasing appreciation of the importance of KLFs in VSMC cell biology. To date, 2 members of this gene family have been shown to critically regulate the SMC response to injury. KLF5 is minimally expressed in the vasculature under basal conditions but is robustly induced after injury. This factor promotes SMC proliferation and, consistent with this observation, hemizygous deficiency of KLF5 results in reduced neointimal formation.8 Similarly, KLF4 is also expressed at low levels in quiescent VSMCs and induced rapidly after injury in VSMCs.9 However, conditional deletion of KLF4 accelerates neointimal formation, suggesting that its postinjury induction is likely a negative feedback response.1 In contrast to KLF4 and KLF5, KLF15 is robustly expressed in VSMCs in vivo under basal conditions and significantly reduced after injury. Given that KLF15 inhibits VSMC proliferation and migration, this high basal expression may serve to prevent unrestrained SMC activation. Taken together, our work, coupled with previous observations, highlights a critical role for the KLF gene family in regulating the vascular response to injury.
Sources of Funding
This study was supported by grants HL086614 (Dr Haldar), HL094660 (Dr Jeyaraj), DK080742 (Dr Gray), HL85816 and HL57506 (Dr Simon), and HL072952, HL075427, HL076754, and HL084154 (Dr Jain) from the National Institutes of Health.
Received on: March 27, 2010; final version accepted on: May 10, 2010.
Yoshida T, Kaestner KH, Owens GK. Conditional deletion of Kruppel-like factor 4 delays downregulation of smooth muscle cell differentiation markers but accelerates neointimal formation following vascular injury. Circ Res. 2008; 102: 1548–1557.
Suzuki T, Sawaki D, Aizawa K, Munemasa Y, Matsumura T, Ishida J, Nagai R. Kruppel-like factor 5 shows proliferation-specific roles in vascular remodeling, direct stimulation of cell growth, and inhibition of apoptosis. J Biol Chem. 2009; 284: 9549–9557.
Grabski AD, Shimizu T, Deou J, Mahoney WM Jr, Reidy MA, Daum G. Sphingosine-1-phosphate receptor-2 regulates expression of smooth muscle alpha-actin after arterial injury. Arterioscler Thromb Vasc Biol. 2009; 29: 1644–1650.
Meredith D, Panchatcharam M, Miriyala S, Tsai YS, Morris AJ, Maeda N, Stouffer GA, Smyth SS. Dominant-negative loss of PPARgamma function enhances smooth muscle cell proliferation, migration, and vascular remodeling. Arterioscler Thromb Vasc Biol. 2009; 29: 465–471.
Bieker JJ. Kruppel-like factors: three fingers in many pies. J Biol Chem. 2001; 276: 34355–34358.
Fisch S, Gray S, Heymans S, Haldar SM, Wang B, Pfister O, Cui L, Kumar A, Lin Z, Sen-Banerjee S, Das H, Petersen CA, Mende U, Burleigh BA, Zhu Y, Pinto YM, Liao R, Jain MK. Kruppel-like factor 15 is a regulator of cardiomyocyte hypertrophy. Proc Natl Acad Sci U S A. 2007; 104: 7074–7079.
Gray S, Feinberg MW, Hull S, Kuo CT, Watanabe M, Sen-Banerjee S, DePina A, Haspel R, Jain MK. The Kruppel-like factor KLF15 regulates the insulin-sensitive glucose transporter GLUT4. J Biol Chem. 2002; 277: 34322–34328.
Shindo T, Manabe I, Fukushima Y, Tobe K, Aizawa K, Miyamoto S, Kawai-Kowase K, Moriyama N, Imai Y, Kawakami H, Nishimatsu H, Ishikawa T, Suzuki T, Morita H, Maemura K, Sata M, Hirata Y, Komukai M, Kagechika H, Kadowaki T, Kurabayashi M, Nagai R. Kruppel-like zinc-finger transcription factor KLF5/BTEB2 is a target for angiotensin II signaling and an essential regulator of cardiovascular remodeling. Nat Med. 2002; 8: 856–863.
Liu Y, Sinha S, McDonald OG, Shang Y, Hoofnagle MH, Owens GK. Kruppel-like factor 4 abrogates myocardin-induced activation of smooth muscle gene expression. J Biol Chem. 2005; 280: 9719–9727.