Nichola Figg

Dialysis Accelerates Medial Vascular Calcification in Part by Triggering Smooth Muscle Cell Apoptosis

Background— Vascular calcification is associated with increased morbidity and mortality in stage V chronic kidney disease, yet its early pathogenesis and initiating mechanisms in vivo remain poorly understood. To address this, we quantified the calcium (Ca) load in arteries from children (10 predialysis, 24 dialysis) and correlated it with clinical, biochemical, and vascular measures. Methods and Results— Vessel Ca load was significantly elevated in both predialysis and dialysis and was correlated with the patients’ mean serum Ca×phosphate product. However, only dialysis patients showed increased carotid intima-media thickness and increased aortic stiffness, and calcification on computed tomography was present in only the 2 patients with the highest Ca loads. Importantly, predialysis vessels appeared histologically intact, whereas dialysis vessels exhibited evidence of extensive vascular smooth muscle cell (VSMC) loss owing to apoptosis. Dialysis vessels also showed increased alkaline phosphatase activity and Runx2 and osterix expression, indicative of VSMC osteogenic transformation. Deposition of the vesicle membrane marker annexin VI and vesicle component mineralization inhibitors fetuin-A and matrix Gla-protein increased in dialysis vessels and preceded von Kossa positive overt calcification. Electron microscopy showed hydroxyapatite nanocrystals within vesicles released from damaged/dead VSMCs, indicative of their role in initiating calcification. Conclusions— Taken together, this study shows that Ca accumulation begins predialysis, but it is the induction of VSMC apoptosis in dialysis that is the key event in disabling VSMC defense mechanisms and leading to overt calcification, eventually with clinically detectable vascular damage. Thus the identification of factors that lead to VSMC death in dialysis will be of prime importance in preventing vascular calcification.

Chronic Apoptosis of Vascular Smooth Muscle Cells Accelerates Atherosclerosis and Promotes Calcification and Medial Degeneration

Vascular smooth muscle cell (VSMC) accumulation is implicated in plaque development. In contrast, VSMC apoptosis is implicated in plaque rupture, coagulation, vessel remodeling, medial atrophy, aneurysm formation, and calcification. Although VSMC apoptosis accompanies multiple pathologies, there is little proof of direct causality, particularly with the low levels of VSMC apoptosis seen in vivo. Using a mouse model of inducible VSMC–specific apoptosis, we demonstrate that low-level VSMC apoptosis during either atherogenesis or within established plaques of apolipoprotein (Apo)E−/− mice accelerates plaque growth by two-fold, associated with features of plaque vulnerability including a thin fibrous cap and expanded necrotic core. Chronic VSMC apoptosis induced development of calcified plaques in younger animals and promoted calcification within established plaques. In addition, VSMC apoptosis induced medial expansion, associated with increased elastic lamina breaks, and abnormal matrix deposition reminiscent of cystic medial necrosis in humans. VSMC apoptosis prevented outward remodeling associated with atherosclerosis resulting in marked vessel stenosis. We conclude that VSMC apoptosis is sufficient to accelerate atherosclerosis, promote plaque calcification and medial degeneration, prevent expansive remodeling, and promote stenosis in atherosclerosis.

Prelamin A Acts to Accelerate Smooth Muscle Cell Senescence and Is a Novel Biomarker of Human Vascular Aging

Background— Hutchinson-Gilford progeria syndrome is a rare inherited disorder of premature aging caused by mutations in LMNA or Zmpste24 that disrupt nuclear lamin A processing, leading to the accumulation of prelamin A. Patients develop severe premature arteriosclerosis characterized by vascular smooth muscle cell (VSMC) calcification and attrition. Methods and Results— To determine whether defective lamin A processing is associated with vascular aging in the normal population, we examined the profile of lamin A expression in normal and aged VSMCs. In vitro, aged VSMCs rapidly accumulated prelamin A coincidently with nuclear morphology defects, and these defects were reversible by treatment with farnesylation inhibitors and statins. In human arteries, prelamin A accumulation was not observed in young healthy vessels but was prevalent in medial VSMCs from aged individuals and in atherosclerotic lesions, where it often colocalized with senescent and degenerate VSMCs. Prelamin A accumulation correlated with downregulation of the lamin A processing enzyme Zmpste24/FACE1, and FACE1 mRNA and protein levels were reduced in response to oxidative stress. Small interfering RNA knockdown of FACE1 reiterated the prelamin A–induced nuclear morphology defects characteristic of aged VSMCs, and overexpression of prelamin A accelerated VSMC senescence. We show that prelamin A acts to disrupt mitosis and induce DNA damage in VSMCs, leading to mitotic failure, genomic instability, and premature senescence. Conclusions— This study shows that prelamin A is a novel biomarker of VSMC aging and disease that acts to accelerate senescence. It therefore represents a novel target to ameliorate the effects of age-induced vascular dysfunction.

Monocyte/Macrophage Suppression in CD11b Diphtheria Toxin Receptor Transgenic Mice Differentially Affects Atherogenesis and Established Plaques

Although monocytes/macrophages are considered important in atherogenesis, their role in established plaques is unclear. For example, macrophage content is associated with plaque instability, but their loss through cell death is observed at sites of plaque rupture. To examine the role of monocytes/macrophages in atherosclerosis, we developed CD11b–diphtheria toxin (DT) receptor (DTR) transgenic mice, whereby administration of DT selectively kills monocytes/macrophages. DT treatment reduced peripheral blood monocytes and tissue macrophages and inhibited macrophage function in CD11b-DTR mice and apolipoprotein E–null (apoE−/−) mice transplanted with CD11b-DTR bone marrow. In atherogenesis experiments, DT markedly reduced plaque development and altered plaque composition, reducing collagen content and necrotic core formation. In mice with established plaques, acute DT treatment induced macrophage apoptosis and reduced macrophage content but did not induce plaque inflammation, thrombosis, or rupture. Furthermore, despite a 50% reduction in monocytes, chronic DT treatment of these mice did not alter plaque extent or composition, most likely because of ongoing recruitment/proliferation of monocytes with recovery of macrophage content. We conclude that monocytes/macrophages are critical to atherogenesis, but established plaques are more resistant to reductions in monocytes.

Chronic Mineral Dysregulation Promotes Vascular Smooth Muscle Cell Adaptation and Extracellular Matrix Calcification

In chronic kidney disease (CKD) vascular calcification occurs in response to deranged calcium and phosphate metabolism and is characterized by vascular smooth muscle cell (VSMC) damage and attrition. To gain mechanistic insights into how calcium and phosphate mediate calcification, we used an ex vivo model of human vessel culture. Vessel rings from healthy control subjects did not accumulate calcium with long-term exposure to elevated calcium and/or phosphate. In contrast, vessel rings from patients with CKD accumulated calcium; calcium induced calcification more potently than phosphate (at equivalent calcium-phosphate product). Elevated phosphate increased alkaline phosphatase activity in CKD vessels, but inhibition of alkaline phosphatase with levamisole did not block calcification. Instead, calcification in CKD vessels most strongly associated with VSMC death resulting from calcium- and phosphate-induced apoptosis; treatment with a pan-caspase inhibitor ZVAD ameliorated calcification. Calcification in CKD vessels was also associated with increased deposition of VSMC-derived vesicles. Electron microscopy confirmed increased deposition of vesicles containing crystalline calcium and phosphate in the extracellular matrix of dialysis vessel rings. In contrast, vesicle deposition and calcification did not occur in normal vessel rings, but we observed extensive intracellular mitochondrial damage. Taken together, these data provide evidence that VSMCs undergo adaptive changes, including vesicle release, in response to dysregulated mineral metabolism. These adaptations may initially promote survival but ultimately culminate in VSMC apoptosis and overt calcification, especially with continued exposure to elevated calcium.

DNA Damage Links Mitochondrial Dysfunction to Atherosclerosis and the Metabolic Syndrome

Rationale: DNA damage is present in both genomic and mitochondrial DNA in atherosclerosis. However, whether DNA damage itself promotes atherosclerosis, or is simply a byproduct of the risk factors that promote atherosclerosis, is unknown. Objective: To examine the effect of DNA damage on atherosclerosis, we studied apolipoprotein (Apo)E−/− mice that were haploinsufficient for the protein kinase ATM (ataxia telangiectasia mutated), which coordinates DNA repair. Methods and Results: ATM+/−/ApoE−/− mice developed accelerated atherosclerosis and multiple features of the metabolic syndrome, including hypertension, hypercholesterolemia, obesity, steatohepatitis, and glucose intolerance. Transplantation with ATM+/+ bone marrow attenuated atherosclerosis but not the metabolic syndrome. ATM+/− smooth muscle cells and macrophages showed increased nuclear DNA damage and defective DNA repair signaling, growth arrest, and apoptosis. Metabolomic screening of ATM+/−/ApoE−/− mouse tissues identified metabolic changes compatible with mitochondrial defects, with increased &bgr;-hydroxybutyrate but reduced lactate, reduced glucose, and alterations in multiple lipid species. ATM+/−/ApoE−/− mouse tissues showed an increased frequency of a mouse mitochondrial “common” deletion equivalent and reduced mitochondrial oxidative phosphorylation. Conclusions: We propose that failure of DNA repair generates defects in cell proliferation, apoptosis, and mitochondrial dysfunction. This in turn leads to ketosis, hyperlipidemia, and increased fat storage, promoting atherosclerosis and the metabolic syndrome. Prevention of mitochondrial dysfunction may represent a novel target in cardiovascular disease.

Endogenous p53 Protects Vascular Smooth Muscle Cells From Apoptosis and Reduces Atherosclerosis in ApoE Knockout Mice

Recent studies have indicated that the tumor suppressor gene p53 limits atherosclerosis in animal models; p53 expression is also increased in advanced human plaques compared with normal vessels, where it may induce growth arrest and apoptosis. However, controversy exists as to the role of endogenous levels of p53 in different cell types that comprise plaques. We examined atherosclerotic plaque development and composition in brachiocephalic arteries and aortas of p53−/−/ApoE−/− mice versus wild type p53 controls. p53−/− mice demonstrated increased aortic plaque formation, with increased rates of cell proliferation and reduced rates of apoptosis in brachiocephalic arteries. Although most proliferating cells were monocyte/macrophages, apoptotic cells were both vascular smooth muscle cells (VSMCs) and macrophages. Transplant of p53 bone marrow to p53−/−/ApoE−/− mice reduced aortic plaque formation and cell proliferation in brachiocephalic plaques, but also markedly reduced apoptosis. To examine p53 regulation of these processes, we studied proliferation and apoptosis in macrophages, bone marrow stromal cells and VSMCs cultured from these mice. Although endogenous p53 promoted apoptosis in macrophages, it protected VSMCs and stromal cells from death, a hitherto unknown function in these cells, in part by inhibiting DNA damage response enzymes. p53 also inhibited stromal cell expression of VSMC markers. We conclude that endogenous levels of p53 protect VSMCs and stromal cells against apoptosis, while promoting apoptosis in macrophages, and protect against atherosclerosis development.

Vascular smooth muscle cell sirtuin 1 protects against DNA damage and inhibits atherosclerosis

Background— Vascular smooth muscle cells (VSMCs) in human atherosclerosis manifest extensive DNA damage and activation of the DNA damage response, a pathway that coordinates cell cycle arrest and DNA repair, or can trigger apoptosis or cell senescence. Sirtuin 1 deacetylase (SIRT1) regulates cell ageing and energy metabolism and regulates the DNA damage response through multiple targets. However, the direct role of SIRT1 in atherosclerosis and how SIRT1 in VSMCs might regulate atherosclerosis are unknown. Methods and Results— SIRT1 expression was reduced in human atherosclerotic plaques and VSMCs both derived from plaques and undergoing replicative senescence. SIRT1 inhibition reduced DNA repair and induced apoptosis, in part, through reduced activation of the repair protein Nijmegen Breakage Syndrome-1 but not p53. Fat feeding reduced SIRT1 and induced DNA damage in VSMCs. VSMCs from mice expressing inactive truncated SIRT1 (&Dgr;ex4) showed increased oxidized low-density lipoprotein–induced DNA damage and senescence. ApoE−/− mice expressing SIRT1&Dgr;ex4 only in smooth muscle cells demonstrated increased DNA damage response activation and apoptosis, increased atherosclerosis, reduced relative fibrous cap thickness, and medial degeneration. Conclusions— SIRT1 is reduced in human atherosclerosis and is a critical regulator of the DNA damage response and survival in VSMCs. VSMC SIRT1 protects against DNA damage, medial degeneration, and atherosclerosis.

Mitochondrial DNA Damage Can Promote Atherosclerosis Independently of Reactive Oxygen Species Through Effects on Smooth Muscle Cells and Monocytes and Correlates With Higher-Risk Plaques in Humans

Background— Mitochondrial DNA (mtDNA) damage occurs in both circulating cells and the vessel wall in human atherosclerosis. However, it is unclear whether mtDNA damage directly promotes atherogenesis or is a consequence of tissue damage, which cell types are involved, and whether its effects are mediated only through reactive oxygen species. Methods and Results— mtDNA damage occurred early in the vessel wall in apolipoprotein E–null (ApoE−/−) mice, before significant atherosclerosis developed. mtDNA defects were also identified in circulating monocytes and liver and were associated with mitochondrial dysfunction. To determine whether mtDNA damage directly promotes atherosclerosis, we studied ApoE−/− mice deficient for mitochondrial polymerase-&ggr; proofreading activity (polG−/−/ApoE−/−). polG−/−/ApoE−/− mice showed extensive mtDNA damage and defects in oxidative phosphorylation but no increase in reactive oxygen species. polG−/−/ApoE−/− mice showed increased atherosclerosis, associated with impaired proliferation and apoptosis of vascular smooth muscle cells, and hyperlipidemia. Transplantation with polG−/−/ApoE−/− bone marrow increased the features of plaque vulnerability, and polG−/−/ApoE−/− monocytes showed increased apoptosis and inflammatory cytokine release. To examine mtDNA damage in human atherosclerosis, we assessed mtDNA adducts in plaques and in leukocytes from patients who had undergone virtual histology intravascular ultrasound characterization of coronary plaques. Human atherosclerotic plaques showed increased mtDNA damage compared with normal vessels; in contrast, leukocyte mtDNA damage was associated with higher-risk plaques but not plaque burden. Conclusions— We show that mtDNA damage in vessel wall and circulating cells is widespread and causative and indicates higher risk in atherosclerosis. Protection against mtDNA damage and improvement of mitochondrial function are potential areas for new therapeutics.

Vascular Smooth Muscle Cell Apoptosis Induces Interleukin-1–Directed Inflammation: Effects of Hyperlipidemia-Mediated Inhibition of Phagocytosis

Rationale: Atherosclerosis is characterized by lipid accumulation in the vessel wall, inflammation, and both macrophage and vascular smooth muscle cell (VSMC) apoptosis. However, whereas VSMC apoptosis in mice with established atherosclerotic plaques or hyperlipidemia increases serum levels of the proatherogenic cytokines monocyte chemotactic protein (MCP)-1, tumor necrosis factor &agr;, and interleukin (IL)-6, the link between hyperlipidemia, apoptosis and inflammation, and the mechanisms by which apoptotic cells promote inflammation in atherosclerosis are unknown. Objective: To determine whether hyperlipidemia affects apoptotic cell clearance, and identify the molecular pathways downstream of VSMC apoptosis that may promote inflammation. Methods and Results: We find that human VSMCs are potent and efficient phagocytes of apoptotic human VSMCs, but phagocytosis is significantly reduced by oxidized low-density lipoprotein in vitro or hyperlipidemia in vivo. Necrotic human aortic VSMCs release IL-1&agr;, which induces IL-6 and MCP-1 production from viable human VSMCs in vitro. In contrast, secondary necrotic VSMCs release both IL-1&agr; and caspase-activated IL-1&bgr;, augmenting IL-6 and MCP-1 production. Conditionally inducing VSMC apoptosis in situ in hyperlipidemic SM22&agr;-hDTR/ApoE−/− mice to levels seen in human plaques increases serum MCP-1, tumor necrosis factor &agr;, and IL-6, which is prevented by blocking IL-1. Conclusions: We conclude that VSMC necrosis releases IL-1&agr;, whereas secondary necrosis of apoptotic VSMCs releases both IL-1&agr; and &bgr;. IL-1 from necrotic VSMCs induces the surrounding viable VSMCs to produce proinflammatory cytokines. Thus, failed clearance of apoptotic VSMCs caused by hyperlipidemia in vivo may promote the increased serum cytokines and chronic inflammation associated with atherosclerosis.