Objective Atherosclerosis is the primary driver of cardiovascular disease, the leading cause of death worldwide. Identification of naturally occurring atheroprotective genes has become a major goal for development of interventions that will limit atheroma progression and associated adverse events. To this end, we have identified SPRR3 (small proline-rich repeat protein 3) as selectively upregulated in vascular smooth muscle cells (VSMCs) of atheroma-bearing arterial tissue versus healthy arterial tissue. In this study, we sought to determine the role of SPRR3 in atheroma pathophysiology. Approach and Results We found that atheroprone ApoE-null mice lacking SPRR3 developed significantly greater atheroma burden. To determine the cellular driver(s) of this increase, we evaluated SPRR3-dependent changes in bone-marrow-derived cells, endothelial cells (ECs), and VSMCs. Bone marrow transplant of SPRR3-expressing cells into SPRR3−/− ApoE−/− recipients failed to rescue atheroma burden. Similarly, ECs did not exhibit a response to SPRR3 loss. However, atheromas from SPRR3-deficient mice exhibited increased TUNEL-positive VSMCs compared with control. Cell death in SPRR3-deficient VSMCs was significantly increased in vitro. Conversely, SPRR3-overexpressing VSMCs exhibited reduced apoptosis compared with control. We also observed a PI3K/Akt-dependent positive association between SPRR3 expression and levels of active Akt in VSMCs. The survival advantage seen in SPRR3-overexpressing VSMCs was abrogated following the addition of a PI3K/Akt pathway inhibitor. Conclusions These results indicate that SPRR3 protects the lesion from VSMC loss by promoting survival signaling in plaque VSMCs, thereby significantly decreasing atherosclerosis progression. As the first identified atheroma-specific VSMC pro-survival factor, SPRR3 represents a potential target for lesion-specific modulation of VSMC survival.

Vascular smooth muscle cells (VSMCs) represent important modulators of plaque stability in advanced lesions. We previously reported that loss of small proline-rich repeat protein 3 (Sprr3), leads to VSMC apoptosis in a PI3K/Akt-dependent manner and accelerates lesion progression. Here, we investigated the role of Sprr3 in modulating plaque stability in hyperlipidemic ApoE-/- mice. We show that loss of Sprr3 increased necrotic core size and reduced cap collagen content of atheromas in brachiocephalic arteries with evidence of plaque rupture and development of intraluminal thrombi. Moreover, Sprr3-/-ApoE-/- mice developed advanced coronary artery lesions accompanied by intraplaque hemorrhage and left ventricle microinfarcts. SPRR3 is known to reduce VSMC survival in lesions by promoting their apoptosis. In addition, we demonstrated that Sprr3-/- VSMCs displayed reduced expression of procollagen in a PI3K/Akt dependent manner. SPRR3 loss also increased MMP gelatinase activity in lesions, and increased MMP2 expression, migration and contraction of VSMCs independently of PI3K/Akt. Consequently, Sprr3 represents the first described VSMC modulator of each of the critical features of cap stability, including VSMC numbers, collagen type I synthesis, and protease activity through Akt dependent and independent pathways.

Atheroma rupture is the leading cause of myocardial infarction. While studies have examined inflammatory cell-mediated effects on plaque vulnerability, less is known about the role of vascular smooth muscle cells (VSMCs) or specific molecular players in the maintenance of atheroma stability. We reported that loss of small proline-rich repeat protein 3 (SPRR3), enriched in atheroma VSMCs, leads to increased VSMC death and significantly accelerates atherosclerosis progression in ApoE -/- mice. Here, we show that loss of SPRR3 promotes features in plaques of brachiocephalic arteries common to unstable lesions, such as increased necrotic core size, reduced cap collagen content, and reduced VSMC content. Moreover, ApoE -/- mice lacking SPRR3 develop coronary artery lesions with advanced features, including intraplaque hemorrhage. In addition, Sprr3 -/- ApoE -/- mice fed a high-fat diet for 6 months develop spontaneous myocardial infarction. In vitro , SPRR3 deficient VSMCs show reduced expression of procollagen type I, an event associated with Akt activation. SPRR3-deficient VSMCs also show increased expression of MMP2 transcripts, and aortic root lesions of Sprr3 -/- ApoE -/- mice have increased gelatinase activity consistent with MMP2 activation. Our data demonstrate that SPRR3 loss in ApoE -/- mice decreases VSMC survival and collagen I synthesis while increasing MMP2 synthesis and activity, resulting in atheroma instability with evidence of downstream myocardial infarction. Taken together the results present the Sprr3 -/- ApoE -/- mouse as an experimental model of plaque rupture. This model will be used for additional experimental studies including in vivo genetic modulation of the Akt pathway as well as in vitro studies to determine phenotypic outcome, i.e. coronary arterial lesions, myocardial infarction, VSMC survival and collagen synthesis. We hope to establish a mechanistic link between altered Akt signaling and matrix integrity in the context of atheroma rupture, as well as potentially use SPRR3 as a molecular marker which could lead to detection of plaque instability as well as therapeutic intervention methodologies.

Cardiovascular disease, the leading cause of death in the United States, is primarily driven by atherosclerosis. In recent years, studies have focused on identification of naturally expressed, atheroprotective genes for use in delaying development of or preventing complications from atherosclerosis. To this end, we previously identified SPRR3 (small proline rich protein 3) as a gene specifically upregulated in vascular smooth muscle cells (VSMCs) of atheroma versus healthy arterial tissue of humans and mice. In the present study, we generated ApoE-null mice lacking SPRR3, which displayed significantly increased atheroma burden compared with ApoE-null controls. To determine the cellular driver(s) of this phenotype, we investigated SPRR3-dependent changes in bone marrow-derived cells, endothelial cells (ECs), and VSMCs. Bone marrow transplant of SPRR3-expressing cells into SPRR3-/-ApoE-/- recipients failed to rescue atheroma burden. Similarly, no change was observed in SPRR3-deficient versus control ECs. However, apoptosis was significantly reduced in SPRR3-overexpressing VSMCs in vitro. A positive association was also observed between SPRR3 expression and PI3K/Akt activity in VSMCs. The SPRR3-dependent survival advantage observed in SPRR3-overexpressing cells was lost following treatment with PI3K/Akt pathway inhibitor. Our data indicate that SPRR3 protects the atheroma from VSMC loss by promoting survival signaling in lesion VSMCs, thereby modulating atherosclerosis development. As the first identified atheroma-specific VSMC pro-survival factor, SPRR3 represents a potential target for lesion-specific modulation of VSMC survival to inhibit plaque progression.