PCSK6-Mediated Regulation of Vascular Remodeling

Helle F. Jørgensen, Martin R. Bennett enetic association studies have significantly enhanced our understanding of vascular disease etiology and offer great promise for identification of candidate drug targets and estimating disease risk.1 In a study recently published in Circulation Research, Rykaczewska et al2 have used the IMPROVE study (Carotid Intima Media Thickness [IMT] and IMT-Progression as Predictors of Vascular Events in a High-Risk European Population; n=3378), AtheroEx- press biobank (n=197), and BiKE (Biobank of Karolinska Endarterectomies) cohorts (n=127) to convincingly dem- onstrate that rs1531817, a noncoding intronic variant in the PCSK6 locus, is associated with decreased PCSK6 (proprotein convertase subtilisin/kexin 6) expression and increased vascular disease severity.

PCSK6 (also known as PACE4, paired basic amino acid– cleaving enzyme 4) belongs to the PCSK family, that activate other proteins by proteolytic cleavage. This family also includes PCSK9, which regulates cholesterol homeostasis by control- ling LDLR (low-density lipoprotein receptor) levels on the cell surface and can be mutated in familial hypercholesterolemia,3 and FURIN/PACE/PCSK3, which has also been both func- tionally and genetically linked to cardiovascular disease.4,5 During development, PCSK6 targets the TGFβ (trans- forming growth factor beta) superfamily member NODAL6 and the protein acts to promote cancer cell proliferation and tumor progression.7,8 Investigation of a possible func- tional importance of PCSK6 in cardiovascular disease was prompted by a previous report of high levels of PCSK6 in atherosclerotic lesions.9 In the current study, PCSK6 was found expressed in αSMA (alpha smooth muscle actin)-positive cells in the fibrous cap of human athero- sclerotic lesions, whereas little PCSK6 was detected in the medial layer of normal arteries. Similar observations were made in a mouse model of atherosclerosis. This suggests that PCSK6 expression is induced in vascular smooth muscle cell (VSMC)-derived atherosclerotic plaque cells.

VSMCs play a pivotal role in the development of athero- sclerosis, where these cells undergo phenotypic modula- tion characterized by loss of contractile gene expression and induction of proliferation, extracellular matrix (ECM) remodeling, and cell migration.10 VSMC phenotypic modu- lation and induction of proliferation are also observed in response to vascular injury and underlies cell accumula- tion leading to restenosis after stenting. PCSK6 protein levels were increased in both human and rodent medial hyperplasia compared with healthy controls and found to co-localize with αSMA-positive cells. This is consistent with the hypothesis that induction of PCSK6 is associated with transition of VSMCs from a quiescent to an activated state. A causative role of PCSK6 induction in vascular dis- ease was suggested by analysis of the injury response in Pcsk6-mutant animals. Carotid artery ligation induces an acute proliferative VSMC response, resulting in for- mation of a VSMC-derived neointimal lesion.11,12 Pcsk6−/− animals had severely reduced neointimal area compared with wild type controls 6 weeks after injury. At this time point, the frequency of medial PCNA-positive cells was increased 2-fold in Pcsk6-mutant arteries. This seems paradoxical; however, it might be explained by the revers- ibility of VSMC phenotypic modulation in this model resulting in a very low replication index even in wild type arteries at this time point. It is, therefore, possible that a proliferation defect would be evident at earlier timepoints.

Key Words: Editorials ■ atherosclerosis ■ cell proliferation ■ homeostasis ■ hyperplasia ■ vascular smooth muscle cell

The opinions expressed in this article are not necessarily those of the editors or of the American Heart Association. Correspondence to: Martin R. Bennett, MB BCh, PhD, Division of Cardiovascular Medicine, University of Cambridge, Box 110, ACCI, where proliferation is more readily detected.11 Alterna- tively, the vastly reduced neointima could be caused by impaired VSMC migration across the internal elastic lamina. Whereas arteries of Pcsk6−/− animals appeared morphologically normal before injury, expression profiling identified reduced levels of genes associated with VSMC contraction, elastic fiber formation, ECM organization, and some cytokine genes in Pcsk6−/− arteries compared with controls. This suggests that maintenance of VSMC phenotype and ECM remodeling is regulated by PCSK6. However, it is important to note that since PCSK6 is deleted in all cells in this model, these effects may not be a consequence of loss of PCSK6 activity in VSMCs.

In contrast, a direct impact of PCSK6 on VSMCs phenotype is suggested by in vitro experiments where depletion of PCSK6 levels in human carotid artery VSMCs reduces expression of contractile VSMC marker genes including Myh11, Acta2, and Synpo2. Conversely, overexpression of PCSK6 was found to reduce wound size using a scratch assay and increase the fraction of PCNA-positive VSMCs after treatment with PDGF-BB, indicating that PCSK6 promotes VSMC proliferation and migration, similar to that observed in cancer cells.7 To understand the molecular mechanisms underlying the effect of PCSK6-mediated regulation of VSMC phe- notype, the authors focused on ECM remodeling. PCSK6 expression was found to correlate with levels of MMPs (matrix metalloproteinases), which have been previously implicated in cardiovascular disease and regulation of VSMC function.13 In particular, PCSK6 levels correlated positively with MMP2 and MMP14. Furthermore, tran- scriptional analysis correlated expression of PCSK6 with a subset of direct or indirect protein interaction partners of MMP2, MMP14, and PCSK6, but only in the acute phase of injury (5 days post ligation).

Interestingly, sev- eral of the PCSK6 co-expressed genes, including Mmp2, Dcn, and Lum, are also part of the transcriptional signa- ture of an atypical VSMC population14 suggesting that PCSK6 might be associated with or promote this state. Proximity-ligation assays were used to show co-location of PCSK6 and MMP14 in tissue sections of injured arteries. Interestingly, MMP14 activity was reduced in Pcsk6−/− arteries and similar to depletion of PCSK6, siRNA targeting of MMP14 also reduced expression of contractile markers. This highlights MMP14 as a pos- sible mediator of PCSK6-dependent VSMC regulation. Investigations into whether the effect of PCSK6 on VSMC behavior in vitro and vascular remodeling in vivo is dependent on MMP14 cleavage are now required to substantiate this idea. In any case, this study provides further evidence that the niche is important for VSMC behavior, and that ECM remodeling is an important factor in cardiovascular disease development. From a clinical perspective, in vivo inhibitors of PCSK6 activity have already been developed and found to have beneficial effects in a cancer model.8 Successful use of

PCSK9 inhibitors in hypercholesteremic patients has shown that clinical benefits can be achieved through targeting of preprotein convertases.15 Interestingly, inhi- bition of FURIN was recently demonstrated to reduce lesion severity in a mouse model of atherosclerosis. All of these studies identify the therapeutic potential of PCSK6-inhibitors in atherosclerosis. However, decipher- ing the role and mechanism of PCSK6 in vascular remod- eling will reveal whether PCSK6-inhibitors similarly could become effective antivascular disease therapies.


From the Division of Cardiovascular Medicine, University of Cambridge, United Kingdom.

Sources of Funding
The authors are funded by the British Heart Foundation (BHF) grants RE/18/1/34212, AVRO01830/RM/17/2/33380, PG/19/6/34153, CH/2000003.


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