Tamoxifen-independent Cre-activity in SMMHC-CreER T2 mice.

Background and aims: Recent technological advances have established vascular smooth muscle cells (SMCs) as central players in atherosclerosis. Increasingly complex genetic mouse models have unveiled that 30-70% of cells in experimentally induced atherosclerotic lesions derive from a handful of medial SMCs, and that these can adopt a broad range of plaque cell phenotypes. Most of these models are based on the SMMHC-CreER T2 mouse line as Cre-driver. Importantly, Cre-activation can be controlled in time (by administration of tamoxifen, TAM), which is critical to avoid unwanted effects of premature recombination events. The aim of this study was to scrutinize an unexpected observation of TAM-independent Cre-activity in this mouse line. Methods: Cre-activity was assessed by PCR in tissues from SMMHC-CreER T2 mice crossed with mice homozygous for loxP-flanked (floxed) exon 4 of Ccn2 (our gene-of-interest), and Ccn2 protein was measured in aortas by targeted mass spectrometry. Results: We observed spontaneous near-complete excision of floxed Ccn2 in aortas from adult mice that were not treated with TAM. As a result, Ccn2 protein was significantly reduced in aortas from these mice, but not to the same extent as TAM-treated littermates. Remarkably, most of the excision was completed in 4-week-old mice. Excision was Cre-dependent, as knockout bands were negligible in heart and liver (dominated by non-SMCs) of these mice, and undetectable in the aorta in the absence of Cre. Conclusion: Our observations warrant caution, and we advocate inclusion of appropriate controls (i.e., TAM-untreated mice) in future studies.

Basement membrane collagen IV deficiency promotes abdominal aortic aneurysm formation.

Abdominal aortic aneurysm (AAA) is a complex disease which is incompletely accounted for. Basement membrane (BM) Collagen IV (COL4A1/A2) is abundant in the artery wall, and several lines of evidence indicate a protective role of baseline COL4A1/A2 in AAA development. Using Col4a1/a2 hemizygous knockout mice (Col4a1/a2+/-, 129Svj background) we show that partial Col4a1/a2 deficiency augmented AAA formation. Although unchallenged aortas were morphometrically and biomechanically unaffected by genotype, explorative proteomic analyses of aortas revealed a clear reduction in BM components and contractile vascular smooth muscle cell (VSMC) proteins, suggesting a central effect of the BM in maintaining VSMCs in the contractile phenotype. These findings were translated to human arteries by showing that COL4A1/A2 correlated to BM proteins and VSMC markers in non-lesioned internal mammary arteries obtained from coronary artery bypass procedures. Moreover, in human AAA tissue, MYH11 (VSMC marker) was depleted in areas of reduced COL4 as assessed by immunohistochemistry. Finally, circulating COL4A1 degradation fragments correlated with AAA progression in the largest Danish AAA cohort, suggesting COL4A1/A2 proteolysis to be an important feature of AAA formation. In sum, we identify COL4A1/A2 as a critical regulator of VSMC phenotype and a protective factor in AAA formation.

A role for collagen type IV in cardiovascular disease?

Over the past decade, studies have repeatedly found single-nucleotide polymorphisms located in the collagen ( COL) 4A1 and COL4A2 genes to be associated with cardiovascular disease (CVD), and the 13q34 locus harboring these genes is one of ~160 genome-wide significant risk loci for coronary artery disease. COL4A1 and COL4A2 encode the α1- and α2-chains of collagen type IV, a major component of basement membranes in various tissues including arteries. Despite the growing body of evidence indicating a role for collagen type IV in CVD, remarkably few studies have aimed to directly investigate such a role. The purpose of this review is to summarize the clinical reports linking 13q34 to coronary artery disease, atherosclerosis, and artery stiffening and to assemble the scattered pieces of evidence from experimental studies based on vascular cells and tissue collectively supporting a role for collagen type IV in atherosclerosis and other macrovascular disease conditions.