Andrew D. Frutkin

Smooth Muscle Cells Give Rise to Osteochondrogenic Precursors and Chondrocytes in Calcifying Arteries

Vascular calcification is a major risk factor for cardiovascular morbidity and mortality. To develop appropriate prevention and/or therapeutic strategies for vascular calcification, it is important to understand the origins of the cells that participate in this process. In this report, we used the SM22-Cre recombinase and Rosa26-LacZ alleles to genetically trace cells derived from smooth muscle. We found that smooth muscle cells (SMCs) gave rise to osteochondrogenic precursor- and chondrocyte-like cells in calcified blood vessels of matrix Gla protein deficient (MGP−/−) mice. This lineage reprogramming of SMCs occurred before calcium deposition and was associated with an early onset of Runx2/Cbfa1 expression and the downregulation of myocardin and Msx2. There was no change in the constitutive expression of Sox9 or bone morphogenetic protein 2. Osterix, Wnt3a, and Wnt7a mRNAs were not detected in either calcified MGP−/− or noncalcified wild-type (MGP+/+) vessels. Finally, mechanistic studies in vitro suggest that Erk signaling might be required for SMC transdifferentiation under calcifying conditions. These results provide strong support for the hypothesis that adult SMCs can transdifferentiate and that SMC transdifferentiation is an important process driving vascular calcification and the appearance of skeletal elements in calcified vascular lesions.

TGF-β1 Limits Plaque Growth, Stabilizes Plaque Structure, and Prevents Aortic Dilation in Apolipoprotein E-Null Mice

Objective—Impairment of transforming growth factor (TGF)-&bgr;1 signaling accelerates atherosclerosis in experimental mice. However, it is uncertain whether increased TGF-&bgr;1 expression would retard atherosclerosis. The role of TGF-&bgr;1 in aneurysm formation is also controversial. We tested whether overexpression of active TGF-&bgr;1 in hyperlipidemic mice affects atherogenesis and aortic dilation. Methods and Results—We generated apolipoprotein E–null mice with transgenes that allow regulated overexpression of active TGF-&bgr;1 in their hearts. Compared to littermate controls, these mice had elevated cardiac and plasma TGF-&bgr;1, less aortic root atherosclerosis (P≤0.002), fewer lesions in the thoracic and abdominal aortae (P≤0.01), less aortic root dilation (P<0.001), and fewer pseudoaneurysms (P=0.02). Mechanistic studies revealed no effect of TGF-&bgr;1 overexpression on plasma lipids or cytokines, or on peripheral lymphoid organ cells. However, aortae of TGF-&bgr;1–overexpressing mice had fewer T-lymphocytes, more collagen, less lipid, lower expression of inflammatory cytokines and matrix metalloproteinase-13, and higher expression of tissue inhibitor of metalloproteinase-2. Conclusions—When overexpressed in the heart and plasma, TGF-&bgr;1 is an antiatherogenic, vasculoprotective cytokine that limits atherosclerosis and prevents aortic dilation. These actions are associated with significant changes in cellularity, collagen and lipid accumulation, and gene expression in the artery wall.

Transforming Growth Factor Beta 1 Induces Neointima Formation Through Plasminogen Activator Inhibitor-1–Dependent Pathways

Objective—The mechanisms through which transforming growth factor (TGF)-&bgr;1 promotes intimal growth, and the pathways through which TGF-&bgr;1 expression is regulated in the artery wall, are incompletely understood. We used a mouse model to investigate mechanisms of TGF-&bgr;1–induced intimal growth. Methods and Results—Adenovirus-mediated overexpression of TGF-&bgr;1 in uninjured carotid arteries of wild-type mice induced formation of a cellular and matrix-rich intima. Intimal growth appeared primarily due to cell migration and matrix accumulation, with only a negligible contribution from cell proliferation. Overexpression of TGF-&bgr;1 also stimulated expression of plasminogen activator inhibitor type 1 (plasminogen activator inhibitor [PAI]-1) in the artery wall. To test the hypothesis that PAI-1 is a critical downstream mediator of TGF-&bgr;1–induced intimal growth, we transduced carotid arteries of PAI-1–deficient (Serpine1−/−) mice with the TGF-&bgr;1–expressing vector. Overexpression of TGF-&bgr;1 in Serpine1−/− arteries did not increase intimal growth, matrix accumulation, cell migration, or proliferation. Moreover, TGF-&bgr;1–transduced arteries of Serpine1−/− mice secreted 6- to 10-fold more TGF-&bgr;1 than did arteries of wild-type mice that were infused with the same concentration of the TGF-&bgr;1–expressing vector. Conclusions—PAI-1 is both a critical mediator of TGF-&bgr;1–induced intimal growth and a key negative regulator of TGF-&bgr;1 expression in the artery wall.

Mechanisms of TGF-β1–Induced Intimal Growth: Plasminogen-Independent Activities of Plasminogen Activator Inhibitor-1 and Heterogeneous Origin of Intimal Cells

Transforming growth factor (TGF)-&bgr;1 is a potent stimulator of intimal growth. We showed previously that TGF-&bgr;1 stimulates intimal growth through early upregulation of plasminogen activator inhibitor-1 (PAI-1) and, subsequently, PAI-1–dependent increases in cell migration and matrix accumulation. We also showed that PAI-1 negatively regulates TGF-&bgr;1 expression in the artery wall. Here we use plasminogen-deficient mice to test whether TGF-&bgr;1–stimulated, PAI-1–dependent intimal growth and PAI-1 suppression of TGF-&bgr;1 expression are mediated through inhibition of plasminogen activation by PAI-1. We also use lineage tracing to investigate the origin of cells in TGF-&bgr;1–induced intimas. Surprisingly, both TGF-&bgr;1–induced, PAI-1–dependent intimal growth and PAI-1 suppression of TGF-&bgr;1 expression are independent of plasminogen. Moreover, approximately 50% of cells that migrate into the intima of TGF-&bgr;1–overexpressing arteries carry a smooth muscle lineage marker, <1% carry a bone marrow lineage marker, and the remaining cells carry neither marker. Therefore, PAI-1 stimulates intimal growth and suppresses TGF-&bgr;1 expression through activities other than inhibition of plasminogen activation. In addition, contrary to widely held models, our results do not support a role for plasmin (or thrombospondin) in TGF-&bgr;1 activation in the artery wall. Further identification of the molecular targets through which PAI-1 stimulates intimal formation and suppresses TGF-&bgr;1 expression in the artery wall may reveal new approaches for inhibiting intimal formation. Our studies also discount bone marrow as an important source from which TGF-&bgr;1 recruits intimal cells and suggest instead that TGF-&bgr;1 induces substantial cell migration either from the adventitia or from an extravascular, but nonhematopoietic source.

Targeted Rearrangement of Floxed Alleles in Smooth Muscle Cells in Vivo

To the Editor: In their biologically and technically important article “Development of a Smooth Muscle-targeted Cre Recombinase Mouse Reveals Novel Insights Regarding Smooth Muscle Myosin Heavy Chain Promoter Regulation”,1 Regan et al reported generation of mice that express cre recombinase from a fragment of the smooth muscle myosin heavy chain (SMMHC) promoter. The biological importance of this article was that it provided strong evidence of either temporally or spatially restricted expression of the SMMHC promoter in smooth muscle cells in vivo. The paper also had two important technical aspects. First, it demonstrated that expression of a SMMHC promoter-cre construct in cre-indicator mice was a more sensitive means of detecting promoter activity than a construct in which the SMMHC promoter expressed lacZ. Second, the SMMHC-cre mice themselves were said to “provide a powerful tool to researchers to study gene function in vascular development/disease by using cre/lox technology to direct smooth-muscle-specific gene activation or inactivation.” Although the SMMHC-cre mice reported by Regan et al have been used by one group for gene activation,2,3 we—as well as other groups that shared their results with us4 …