CDKN2B Regulates TGF β Signaling and Smooth Muscle Cell Investment of Hypoxic Neovessels

Abstract

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.

Deficient CDKN2B Expression

“…6 In extending this knowledge, the Leeper group at Stanford utilized CDKN2B−/− mice to demonstrate increased SMC apoptosis and decreased SMC phagocytic clearance in mouse models of aneurysm and atherosclerosis, respectively. 5,6 In Nanda et al, 8 published in this issue of Circulation Research , the Leeper group presents exciting evidence for a role of CDKN2B in PAD pathogenesis.…”

“…Increased microvessel density within atherosclerotic lesions has been linked to increased rates of intraplaque inflammation, hemorrhage and rupture. 9 This human observation by Nanda et al 8 suggests that it may not only be the presence of endothelial cells (ECs) within the plaque that is important but also whether or not the ECs are invested by perivascular cells, be it smooth muscle cells (SMCs) or pericytes. Throughout the study, the authors use α-SMA staining, which cannot distinguish between SMCs and pericytes, as pericytes can also express α-SMA, particularly in an inflammatory setting.…”

“…It is interesting to note that roughly half of PAD patients, despite having a reduced ankle-brachial index (ABI), report no symptoms, suggesting their ability to mount an effective neovascular response. 11 In light of the findings in Nanda et al, 8 could reduced CDKN2B expression lead to impaired functional neovascularization and therefore worse outcomes for PAD patients? Conversely, if an iliac or proximal femoral artery occlusion occurs in a patient, can robust CDKN2B expression allow sufficient revascularization so that the PAD becomes undiagnosed?…”

“…6–8 Although aneurysm and atherosclerotic plaque development are often associated with defective SMC function, the role of the SMC in neovessel formation/angiogenesis is clearly understudied. As a point of clarity, the authors occasionally use the term ‘angiogenesis’ as a more general term for neovascularization, or new vessel formation.…”

“…The fact that Nanda et al 8 has uncovered a novel mechanism whereby reduced CDKN2B expression impacts both atherosclerotic and non-atherosclerotic disease, via inhibition of neovessel maturation, is exciting, particularly in light of recent GWASs. Several studies have linked SNPs within the 9p21 locus with PAD independent of atherosclerotic risk factors.…”