CDKN2B Regulates TGFβ Signaling and Smooth Muscle Cell Investment of Hypoxic Neovessels
Rationale: Genetic variation at the chromosome 9p21 cardiovascular risk locus has been associated with peripheral artery disease (PAD), but its mechanism remains unknown.
Objective: To determine whether this association is secondary to an increase in atherosclerosis, or is the result of a separate angiogenesis-related mechanism.
Methods and Results: Quantitative evaluation of human vascular samples revealed that carriers of the 9p21 risk allele possess a significantly higher burden of immature intraplaque microvessels than carriers of the ancestral allele, irrespective of lesion size or patient comorbidity. To determine whether aberrant angiogenesis also occurs under non-atherosclerotic conditions, we performed femoral artery ligation surgery in mice lacking the 9p21 candidate gene, Cdkn2b. These animals developed advanced hind-limb ischemia and digital auto-amputation, secondary to a defect in the capacity of the Cdkn2b-deficient smooth muscle cell (SMC) to support the developing neovessel. Microarray studies identified impaired TGFβ signaling in cultured CDKN2B-deficient cells, as well as TGFβ1 upregulation in the vasculature of 9p21 risk allele carriers. Molecular signaling studies indicated that loss of CDKN2B impairs the expression of the inhibitory factor, SMAD-7, which promotes downstream TGFβ activation. Ultimately, this manifests in the upregulation of a poorly studied effector molecule, TGFβ1-induced-1, which is a TGFβ-'rheostat' known to have antagonistic effects on the EC and SMC. Dual knockdown studies confirmed the reversibility of the proposed mechanism, in vitro.
Conclusions: These results suggest that loss of CDKN2B may not only promote cardiovascular disease through the development of atherosclerosis, but may also impair TGFβ signaling and hypoxic neovessel maturation.
- Received October 27, 2015.
- Revision received November 17, 2015.
- Accepted November 20, 2015.