Downregulation of cytoskeletal markers LMOD1, SYNPO2, PDLIM7,

disease SMCs likely become overloaded, undergo apoptosis and, due to defective efferocytosis, and also secondary necrosis, ultimately contribute to the growing necrotic core.

This has been validated by Shankman et al. showing that SMC-specific loss of Klf4 and associated >50% reduction specifically in the number of Lgals3+ SMCs resulted in a significant decrease in the overall lesion size with a concomitant increase in the indices of lesion stability¹⁵⁰. Whether loss of BCLAF1 would yield similar results is yet to be corroborated.

4.2.2 Downregulation of cytoskeletal markers LMOD1, SYNPO2, PDLIM7,

identified muscle contraction, muscle development and actin cytoskeleton among the most downregulated pathways in atherosclerotic plaques compared to normal arteries, while LMOD1, SYNPO2, PLN, PDLIM7 and SYNM (synemin), previously not associated with cardiovascular disease, were among the most suppressed transcripts both in late-stage human plaques and in lesions from patients with cerebrovascular symptoms (Figure 19A). Strong positive correlations have been found for all of the repressed molecules with classical contractility markers such as CNN1, MYH11 and ACTA2, with the link to SMCs further confirmed by all these proteins being abundantly expressed in normal arteries and directly or indirectly associated with actomyosin cytoskeleton. Intriguingly, stainings performed in aortic lesions from different stages of atheroprogression, as well as in late-stage plaques revealed various levels of sensitivity of these markers.

While SYNPO2, LMOD1 and PLN represented profiles similar to MYH11 and were mostly absent already from stage I adaptive intimal thickening, PDLIM7, like less sensitive SMC markers CNN1 and SMA, reappeared at stage III pathological intimal thickening and was expressed both in the media and weakly also in SMCs forming the fibrous cap. SYNM, with an intermediate sensitivity profile, reappeared from stage III in subintimal SMCs while it was mostly undetectable in SMA+ cells within the fibrous cap. Interestingly, while in human and rat intimal hyperplasia initial downregulation of both classical contractility markers as well as LMOD1, SYNPO2, PDLIM7, PLN and SYNM was reversible and followed by reacquisition of contractile phenotype, majority of the proteins remained repressed in the atherosclerotic fibrous cap from human lesions (Figure 19B). This can be explained by either the existence of the heterogeneous population of SMCs contributing to the lesion development and progression or presence of local environmental cues that enforce sustained downregulation of these markers.

In favor of the latter concept, the exposure of SMCs to major atherosclerosis-relevant stimuli, including proinflammatory growth factors, shear stress and oxLDL, resulted in a significant downregulation of all of our new proposed markers (Figure 20A-C). Functionally, in vitro silencing of PDLIM7, which we have shown in this study to be genetically associated with altered cIMT phenotypes in high-risk individuals⁷⁵ and also previously reported to represent

Figure 19. LMOD1, SYNPO2, SYNM, PDLIM7 and PLN are localized to SMCs and repressed in atherosclerosis and intimal hyperplasia. LMOD1, SYNPO2, SYNM, PDLIM7 and PLN were significantly downregulated in carotid plaques and lesions from symptomatic patients (A). By IHC, PDLIM7 and LMOD1 were abundantly expressed in SMCs in the normal arteries, while their expression pattern differed in the diseased tissue. LMOD1, as a sensitive marker, was lost in plaques and intimal hyperplasia. On the contrary, PDLIM7 remained strongly expressed in subintimal SMA+ in the periphery of the plaque, as well as in the intimal hyperplasia (B). Adapted from Study I, Figures 1 and 2.

one of the key nodes in the plaque functional protein networks⁴¹², led to downregulation of other both established (MYH11, ACTA2) as well as novel cytoskeleton markers (SYNPO2, LMOD1, PLN), impaired adhesion and spreading, as well as increased proliferation of SMCs (Figure 20D and E), suggesting an important structural and mechanistic role of PDLIM7 for maintenance of differentiated phenotype of these cells.

Since our publication in 2016, several studies have supported our findings and further explored the role of LMOD1, SYNPO2 and SYNM. SYNM intermediate filament protein has been confirmed to localize exclusively to muscle tissue and identified as an SRF-independent downstream target of MYOCD⁴²³. SYNM-deficient mice displayed myopathic changes leading to impaired response to injury⁴²⁴, likely due to the imbalance in the regulation of cell quiescence/self-renewal⁴²⁵. Similarly, SYNPO2 and LMOD1 have been reported to be under the regulation of MYOCD and myocardin-related transcription factors (MRTFs), however here both markers of contractile SMCs were shown to be SRF-dependent^{223, 426}. Of interest, LMOD1 knockdown has been associated with impaired human coronary SMCs contraction, as well as increased proliferation rate and migratory capacity⁴²⁷, therefore supporting our claims that this molecule is of importance for the preservation of differentiated phenotype by SMCs.

Of particular interest, in relation to Study IV presented in this Ph.D. thesis, PDLIM7 and SYNM have been predicted as FOXC1 downstream targets, which was further confirmed by these markers being downregulated in response to FOXC1 silencing. In addition, although there was no evidence for the direct FOXC1 regulation of SYNPO2 and LMOD1, both transcripts were repressed in response to FOXC1 knockdown, further strengthening our notion of FOXC1 being the key upstream transcription factor in regulation of SMC quiescence.

Altogether, there is a sufficient body of evidence showing that LMOD1, SYNPO2, PDLIM7, PLN and SYNM are of crucial importance for the physiological contractile function of the healthy SMCs. However, in the light of our results showing that in response to injury and/or atherogenesis all of these markers are early on strongly repressed, the relevant question

Figure 20. Novel SMC markers are repressed in response to pro-atherogenic stimuli. PDLIM7 downregulation results in repression of typical SMC markers and their increased proliferation. LMOD1 and PDLIM7 were significantly downregulated upon exposure to INFg, oxLDL and shear stress (A-C).

PDLIM7 silencing resulted in repression of classical contractility markers and increased proliferation of SMCs (D-E). Adapted from Study I, Figures 6 and 8.

appears: what could it mean for the vessel wall homeostasis? One possible explanation could be that cells lose their contractility and actomyosin cytoskeleton-related proteins, as their contractile function is no longer a priority in the injured/diseased vessel. As a result, they acquire novel features, likely evolutionary advantageous for the cell survival and preserved tissue integrity under the new conditions in the disease vessel wall. As an alternative to apoptosis, SMCs adopt to the changing environmental cues and phenotypically modulate in order to counteract and/or repair the changes in their surroundings. Importantly, while relatively fast abolition of these changes allows for quick reacquisition of classical contractile features, chronic disease-related alterations enforce preservation of SMC synthetic or trans-differentiated phenotype, which depending on the nature of transition, may be either beneficial or detrimental for the lesion stabilization.