Caspase-3 Deletion Promotes Necrosis in Atherosclerotic Plaques of ApoE Knockout Mice

Apoptosis of macrophages and vascular smooth muscle cells (VSMCs) in advanced atherosclerotic plaques contributes to plaque progression and instability. Caspase-3, a key executioner protease in the apoptotic pathway, has been identified in human and mouse atherosclerotic plaques but its role in atherogenesis is not fully explored. We therefore investigated the impact of caspase-3 deletion on atherosclerosis by crossbreeding caspase-3 knockout (Casp3 −/− ) mice with apolipoprotein E knockout (ApoE −/− ) mice. Bone marrow-derived macrophages and VSMCs isolated from Casp3 −/− ApoE −/− mice were resistant to apoptosis but showed increased susceptibility to necrosis. However, caspase-3 deficiency did not sensitize cells to undergo RIP1-dependent necroptosis. To study the effect on atherosclerotic plaque development, Casp3 +/+ ApoE −/− and Casp3 −/− ApoE −/− mice were fed a western-type diet for 16 weeks. Though total plasma cholesterol, triglycerides, and LDL cholesterol levels were not altered, both the plaque size and percentage necrosis were significantly increased in the aortic root of Casp3 −/− ApoE −/− mice as compared to Casp3 +/+ ApoE −/− mice. Macrophage content was significantly decreased in plaques of Casp3 −/− ApoE −/− mice as compared to controls, while collagen content and VSMC content were not changed. To conclude, deletion of caspase-3 promotes plaque growth and plaque necrosis in ApoE −/− mice, indicating that this antiapoptotic strategy is unfavorable to improve atherosclerotic plaque stability

Impact of myeloid RIPK1 gene deletion on atherogenesis in ApoE-deficient mice

Background and aims: Targeting macrophage death is a promising strategy for stabilizing atherosclerotic plaques. Recently, necroptosis was identified as a form of regulated necrosis in atherosclerosis. Receptor-interacting serine/threonine-protein kinase (RIPK)1 is an upstream regulator of RIPK3, which is a crucial kinase for necroptosis induction. We aimed to investigate the impact of myeloid-specific RIPK1 gene deletion on atherogenesis. Methods: RIPK1(F/F)LysM-Cre(+)ApoE(-/-) and RIPK1(+/+)LysM-Cre(+)ApoE(-/-) mice were fed a western-type diet (WD) for 16 or 24 weeks to induce plaque formation. Results: After 16 weeks WD, plaque area and percentage necrosis in RIPK1(F/F)LysM-Cre(+)ApoE(-/-) mice were significantly decreased as compared to plaques of RIPK1(+/+)LysM-Cre(+)ApoE(-/-) mice. Moreover, plaques of RIPK1(F/F)LysM-Cre(+)ApoE(-/-) mice showed more apoptosis and a decreased macrophage content. After 24 weeks WD, plaque size and percentage necrosis were no longer different between the two groups. Free apoptotic cells strongly accumulated in plaques of RIPK1(F/F)LysM-Cre(+)ApoE(-/-) mice. In addition to apoptosis, necroptosis was upregulated in plaques of RIPK1(F/F)LysM-Cre(+)ApoE(-/-) mice. In vitro, TNF-alpha triggered apoptosis in RIPK1(F/F)LysM-Cre(+)ApoE(-/-), but not in RIPK1(+/+)LysM-Cre(+)ApoE(-/-) macrophages. Moreover, RIPK1(F/F)LysM-Cre(+)ApoE(-/-) macrophages were not protected against RIPK3-dependent necroptosis Conclusions: The impact of myeloid RIPK1 gene deletion depends on the stage of atherogenesis. At 16 weeks WD, myeloid RIPK1 gene deletion resulted in increased apoptosis, thereby slowing down plaque progression. However, despite decreased macrophage content, plaque and necrotic core size were no longer reduced after 24 weeks of WD, most likely due to the accumulation of free apoptotic and necroptotic cells

Defective autophagy in vascular smooth muscle cells accelerates senescence and promotes neointima formation and atherogenesis

<p>Autophagy is triggered in vascular smooth muscle cells (VSMCs) of diseased arterial vessels. However, the role of VSMC autophagy in cardiovascular disease is poorly understood. Therefore, we investigated the effect of defective autophagy on VSMC survival and phenotype and its significance in the development of postinjury neointima formation and atherosclerosis. Tissue-specific deletion of the essential autophagy gene <i>Atg7</i> in murine VSMCs (<i>atg7</i>^{<i>−/−</i>} VSMCs) caused accumulation of SQSTM1/p62 and accelerated the development of stress-induced premature senescence as shown by cellular and nuclear hypertrophy, CDKN2A-RB-mediated G₁ proliferative arrest and senescence-associated GLB1 activity. Transfection of SQSTM1-encoding plasmid DNA in <i>Atg7</i>^<i>+/+</i> VSMCs induced similar features, suggesting that accumulation of SQSTM1 promotes VSMC senescence. Interestingly, <i>atg7</i>^{<i>−/−</i>} VSMCs were resistant to oxidative stress-induced cell death as compared to controls. This effect was attributed to nuclear translocation of the transcription factor NFE2L2 resulting in upregulation of several antioxidative enzymes. In vivo, defective VSMC autophagy led to upregulation of MMP9, TGFB and CXCL12 and promoted postinjury neointima formation and diet-induced atherogenesis. Lesions of VSMC-specific <i>atg7</i> knockout mice were characterized by increased total collagen deposition, nuclear hypertrophy, CDKN2A upregulation, RB hypophosphorylation, and GLB1 activity, all features typical of cellular senescence. To conclude, autophagy is crucial for VSMC function, phenotype, and survival. Defective autophagy in VSMCs accelerates senescence and promotes ligation-induced neointima formation and diet-induced atherogenesis, implying that autophagy inhibition as therapeutic strategy in the treatment of neointimal stenosis and atherosclerosis would be unfavorable. Conversely, stimulation of autophagy could be a valuable new strategy in the treatment of arterial disease.</p

Telomere damage promotes vascular smooth muscle cell senescence and immune cell recruitment after vessel injury.

Accumulation of vascular smooth muscle cells (VSMCs) is a hallmark of multiple vascular pathologies, including following neointimal formation after injury and atherosclerosis. However, human VSMCs in advanced atherosclerotic lesions show reduced cell proliferation, extensive and persistent DNA damage, and features of premature cell senescence. Here, we report that stress-induced premature senescence (SIPS) and stable expression of a telomeric repeat-binding factor 2 protein mutant (TRF2T188A) induce senescence of human VSMCs, associated with persistent telomeric DNA damage. VSMC senescence is associated with formation of micronuclei, activation of cGAS-STING cytoplasmic sensing, and induction of multiple pro-inflammatory cytokines. VSMC-specific TRF2T188A expression in a multicolor clonal VSMC-tracking mouse model shows no change in VSMC clonal patches after injury, but an increase in neointima formation, outward remodeling, senescence and immune/inflammatory cell infiltration or retention. We suggest that persistent telomere damage in VSMCs inducing cell senescence has a major role in driving persistent inflammation in vascular disease

Long-Term Depletion of Conventional Dendritic Cells Cannot Be Maintained in an Atherosclerotic Zbtb46-DTR Mouse Model

<div><p>Background and aims</p><p>Increased evidence suggests a pro-atherogenic role for conventional dendritic cells (cDC). However, due to the lack of an exclusive marker for cDC, their exact contribution to atherosclerosis remains elusive. Recently, a unique transcription factor was described for cDC, namely <i>Zbtb46</i>, enabling us to selectively target this cell type in mice.</p><p>Methods</p><p>Low-density lipoprotein receptor-deficient (<i>Ldlr</i>^<i>-/-</i>) mice were transplanted with bone marrow from <i>Zbtb46</i>-diphtheria toxin receptor (DTR) transgenic mice following total body irradiation. <i>Zbtb46</i>-DTR→<i>Ldlr</i>^<i>-/-</i> chimeras were fed a Western-type diet for 18 weeks while cDC were depleted by administering diphtheria toxin (DT).</p><p>Results</p><p>Although we confirmed efficient direct induction of cDC death <i>in vitro</i> and <i>in vivo</i> upon DT treatment of <i>Zbtb46</i>-DTR mice, advanced atherosclerotic plaque size and composition was not altered. Surprisingly, however, analysis of <i>Zbtb46</i>-DTR→<i>Ldlr</i>^<i>-/-</i> chimeras showed that depletion of cDC was not sustained following 18 weeks of DT treatment. In contrast, high levels of anti-DT antibodies were detected.</p><p>Conclusions</p><p>Because of the observed generation of anti-DT antibodies and consequently the partial depletion of cDC, no clear decision can be taken on the role of cDC in atherosclerosis. Our results underline the unsuitability of <i>Zbtb46</i>-DTR→<i>Ldlr</i>^-/- mice for studying the involvement of cDC in maintaining the disease process of atherosclerosis, as well as of other chronic inflammatory diseases.</p></div

Weight and plasma characteristics from control and DT-treated <i>Zbtb46</i>-DTR→<i>Ldlr</i>^-/- mice.

<p>Weight and plasma characteristics from control and DT-treated <i>Zbtb46</i>-DTR→<i>Ldlr</i>^-/- mice.</p

Primer sequences for determination of chimerism.

<p>Primer sequences for determination of chimerism.</p

Chronic DT administration does not affect atherosclerotic plaque size and composition in <i>Zbtb46</i>-DTR→<i>Ldlr</i>^<i>-/-</i> mice.

<p>Representative images and quantification of (A) atherosclerotic lesion size (Oil Red O⁺ area), (B) vascular smooth muscle cells (α-SMA staining), (C) collagen (Sirius Red staining), (D) apoptosis (cleaved caspase-3 staining), and (E) CD11c⁺ cells in aortic root cryosections of control and DT-treated <i>Zbtb46</i>-DTR→<i>Ldlr</i>^<i>-/-</i> mice (n = 14–19); Univariate analyses were performed for plaque area and composition of aortic root sections. Data that failed the Levene's test of homogeneity of variances were mathematically transformed before statistical analysis was performed.</p