Jack M. Heath

Smooth Muscle Cell–Specific Runx2 Deficiency Inhibits Vascular Calcification

Rationale: Vascular calcification is a hallmark of atherosclerosis, a major cause of morbidity and mortality in the United States. We have previously reported that the osteogenic transcription factor Runx2 is an essential and sufficient regulator of calcification of vascular smooth muscle cells (VSMC) in vitro. Objective: To determine the contribution of osteogenic differentiation of VSMC to the pathogenesis of vascular calcification and the function of VSMC-derived Runx2 in regulating calcification in vivo. Methods and Results: SMC-specific Runx2-deficient mice, generated by breeding SM22&agr;-Cre mice with the Runx2 exon 8 floxed mice, exhibited normal aortic gross anatomy and expression levels of SMC-specific marker genes. Runx2 deficiency did not affect basal SMC markers, but inhibited oxidative stress-reduced expression of SMC markers. High-fat-diet-induced vascular calcification in vivo was markedly inhibited in the Runx2-deficient mice in comparison with their control littermates. Runx2 deficiency inhibited the expression of receptor activator of nuclear factor &kgr;B ligand, which was accompanied by decreased macrophage infiltration and formation of osteoclast-like cells in the calcified lesions. Coculture of VSMC with bone marrow–derived macrophages demonstrated that the Runx2-deficient VSMC failed to promote differentiation of macrophages into osteoclast-like cells. Conclusions: These data have determined the importance of osteogenic differentiation of VSMC in the pathogenesis of vascular calcification in mice and defined the functional role of SMC-derived Runx2 in regulating vascular calcification and promoting infiltration of macrophages into the calcified lesion to form osteoclast-like cells. Our studies suggest that the development of vascular calcification is coupled with the formation of osteoclast-like cells, paralleling the bone remodeling process.

Runx2-Upregulated Receptor Activator of Nuclear Factor κB Ligand in Calcifying Smooth Muscle Cells Promotes Migration and Osteoclastic Differentiation of Macrophages

Objective—Clinical and experimental studies demonstrate the important roles of vascular smooth muscle cells (VSMC) in the pathogenesis of atherosclerosis. We have previously determined that the osteogenic transcription factor Runx2 is essential for VSMC calcification. The present study characterized Runx2-regulated signals and their potential roles in vascular calcification. Methods and Results—In vivo studies with atherogenic apolipoprotein E−/− mice demonstrated that increased oxidative stress was associated with upregulation of Runx2 and receptor activator of nuclear factor &kgr;B ligand (RANKL), which colocalized in the calcified atherosclerotic lesions and were juxtaposed to infiltrated macrophages and osteoclast-like cells that are positively stained for an osteoclast marker, tartrate-resistant acid phosphatase. Mechanistic studies using RNA interference, a luciferase reporter system, chromatin immunoprecipitation, and electrophoretic mobility shift assays indicated that Runx2 regulated the expression of RANKL via a direct binding to the 5′-flanking region of the RANKL. Functional characterization revealed that RANKL did not induce VSMC calcification, nor was RANKL required for oxidative stress–induced VSMC calcification. Using a coculture system, we demonstrated that VSMC-expressed RANKL induced migration as well as differentiation of bone marrow-derived macrophages into multinucleated, tartrate-resistant acid phosphatase–positive osteoclast-like cells. These effects were inhibited by the RANKL antagonist osteoprotegerin and with VSMC deficient in Runx2 or RANKL. Conclusion—We demonstrate that Runx2 directly binds to the promoter and controls the expression of RANKL, which mediates the crosstalk between calcifying VSMC and migration and differentiation of macrophages into osteoclast-like cells in the atherosclerotic lesions. Our studies provide novel mechanistic insights into the regulation and function of VSMC-derived RANKL in the pathogenesis of atherosclerosis and vascular calcification.

Activation of AKT by O-Linked N-Acetylglucosamine Induces Vascular Calcification in Diabetes Mellitus

Rationale: Vascular calcification is a serious cardiovascular complication that contributes to the increased morbidity and mortality of patients with diabetes mellitus. Hyperglycemia, a hallmark of diabetes mellitus, is associated with increased vascular calcification and increased modification of proteins by O-linked N-acetylglucosamine (O-GlcNAcylation). Objective: We sought to determine the role of protein O-GlcNAcylation in regulating vascular calcification and the underlying mechanisms. Methods and Results: Low-dose streptozotocin-induced diabetic mice exhibited increased aortic O-GlcNAcylation and vascular calcification, which was also associated with impaired aortic compliance in mice. Elevation of O-GlcNAcylation by administration of Thiamet-G, a potent inhibitor for O-GlcNAcase that removes O-GlcNAcylation, further accelerated vascular calcification and worsened aortic compliance of diabetic mice in vivo. Increased O-GlcNAcylation, either by Thiamet-G or O-GlcNAcase knockdown, promoted calcification of primary mouse vascular smooth muscle cells. Increased O-GlcNAcylation in diabetic arteries or in the O-GlcNAcase knockdown vascular smooth muscle cell upregulated expression of the osteogenic transcription factor Runx2 and enhanced activation of AKT. O-GlcNAcylation of AKT at two new sites, T430 and T479, promoted AKT phosphorylation, which in turn enhanced vascular smooth muscle cell calcification. Site-directed mutation of AKT at T430 and T479 decreased O-GlcNAcylation, inhibited phosphorylation of AKT at S473 and binding of mammalian target of rapamycin complex 2 to AKT, and subsequently blocked Runx2 transactivity and vascular smooth muscle cell calcification. Conclusions: O-GlcNAcylation of AKT at 2 new sites enhanced AKT phosphorylation and activation, thus promoting vascular calcification. Our studies have identified a novel causative effect of O-GlcNAcylation in regulating vascular calcification in diabetes mellitus and uncovered a key molecular mechanism underlying O-GlcNAcylation–mediated activation of AKT.

Inhibition of FOXO1/3 Promotes Vascular Calcification

Objective—Vascular calcification is a characteristic feature of atherosclerosis, diabetes mellitus, and end-stage renal disease. We have demonstrated that activation of protein kinase B (AKT) upregulates runt-related transcription factor 2 (Runx2), a key osteogenic transcription factor that is crucial for calcification of vascular smooth muscle cells (VSMC). Using mice with SMC-specific deletion of phosphatase and tensin homolog (PTEN), a major negative regulator of AKT, the present studies uncovered a novel molecular mechanism underlying PTEN/AKT/FOXO (forkhead box O)-mediated Runx2 upregulation and VSMC calcification. Approach and Results—SMC-specific PTEN deletion mice were generated by crossing PTEN floxed mice with SM22&agr;-Cre transgenic mice. The PTEN deletion resulted in sustained activation of AKT that upregulated Runx2 and promoted VSMC calcification in vitro and arterial calcification ex vivo. Runx2 knockdown did not affect proliferation but blocked calcification of the PTEN-deficient VSMC, suggesting that PTEN deletion promotes Runx2-depedent VSMC calcification that is independent of proliferation. At the molecular level, PTEN deficiency increased the amount of Runx2 post-transcriptionally by inhibiting Runx2 ubiquitination. AKT activation increased phosphorylation of FOXO1/3 that led to nuclear exclusion of FOXO1/3. FOXO1/3 knockdown in VSMC phenocopied the PTEN deficiency, demonstrating a novel function of FOXO1/3, as a downstream signaling of PTEN/AKT, in regulating Runx2 ubiquitination and VSMC calcification. Using heterozygous SMC-specific PTEN-deficient mice and atherogenic ApoE−/− mice, we further demonstrated AKT activation, FOXO phosphorylation, and Runx2 ubiquitination in vascular calcification in vivo. Conclusions—Our studies have determined a new causative effect of SMC-specific PTEN deficiency on vascular calcification and demonstrated that FOXO1/3 plays a crucial role in PTEN/AKT-modulated Runx2 ubiquitination and VSMC calcification.

Redox signaling in cardiovascular pathophysiology: A focus on hydrogen peroxide and vascular smooth muscle cells

Oxidative stress represents excessive intracellular levels of reactive oxygen species (ROS), which plays a major role in the pathogenesis of cardiovascular disease. Besides having a critical impact on the development and progression of vascular pathologies including atherosclerosis and diabetic vasculopathy, oxidative stress also regulates physiological signaling processes. As a cell permeable ROS generated by cellular metabolism involved in intracellular signaling, hydrogen peroxide (H2O2) exerts tremendous impact on cardiovascular pathophysiology. Under pathological conditions, increased oxidase activities and/or impaired antioxidant systems results in uncontrolled production of ROS. In a pro-oxidant environment, vascular smooth muscle cells (VSMC) undergo phenotypic changes which can lead to the development of vascular dysfunction such as vascular inflammation and calcification. Investigations are ongoing to elucidate the mechanisms for cardiovascular disorders induced by oxidative stress. This review mainly focuses on the role of H2O2 in regulating physiological and pathological signals in VSMC.

Vascular Semaphorin 7A Upregulation by Disturbed Flow Promotes Atherosclerosis Through Endothelial β1 Integrin

Objective— Accumulating evidence suggests a role of semaphorins in vascular homeostasis. Here, we investigate the role of Sema7A (semaphorin 7A) in atherosclerosis and its underlying mechanism. Approach and Results— Using genetically engineered Sema7A−/−ApoE−/− mice, we showed that deletion of Sema7A attenuates atherosclerotic plaque formation primarily in the aorta of ApoE−/− mice on a high-fat diet. A higher level of Sema7A in the atheroprone lesser curvature suggests a correlation of Sema7A with disturbed flow. This notion is supported by elevated Sema7A expression in human umbilical venous endothelial cells either subjected to oscillatory shear stress or treated with the PKA (protein kinase A)/CREB (cAMP response element-binding protein) inhibitor H89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide·2HCl hydrate). Further studies using the partial carotid artery ligation model showed that disturbed flow in the left carotid artery of Sema7A+/+ApoE−/− mice promoted the expression of endothelial Sema7A and cell adhesion molecules, leukocyte adhesion, and plaque formation, whereas such changes were attenuated in Sema7A−/−ApoE−/− mice. Further studies showed that blockage of &bgr;1 integrin, a known Sema7A receptor, or inhibition of FAK (focal adhesion kinase), MEK1/2 (mitogen-activated protein kinase kinase 1/2), or NF-&kgr;B (nuclear factor-&kgr;B) significantly reduced the expression of cell adhesion molecules and THP-1 (human acute monocytic leukemia cell line) monocyte adhesion in Sema7A-overexpressing human umbilical venous endothelial cells. Studies using chimeric mice suggest that vascular, most likely endothelial, Sema7A plays a major role in atherogenesis. Conclusions— Our findings indicate a significant role of Sema7A in atherosclerosis by mediating endothelial dysfunction in a &bgr;1 integrin–dependent manner.

Activation of AKT by O-GlcNAcylation Induces Vascular Calcification in Diabetes

Rationale: Vascular calcification is a serious cardiovascular complication that contributes to the increased morbidity and mortality of patients with diabetes mellitus. Hyperglycemia, a hallmark of diabetes mellitus, is associated with increased vascular calcification and increased modification of proteins by O-linked N-acetylglucosamine (O-GlcNAcylation). Objective: We sought to determine the role of protein O-GlcNAcylation in regulating vascular calcification and the underlying mechanisms. Methods and Results: Low-dose streptozotocin-induced diabetic mice exhibited increased aortic O-GlcNAcylation and vascular calcification, which was also associated with impaired aortic compliance in mice. Elevation of O-GlcNAcylation by administration of Thiamet-G, a potent inhibitor for O-GlcNAcase that removes O-GlcNAcylation, further accelerated vascular calcification and worsened aortic compliance of diabetic mice in vivo. Increased O-GlcNAcylation, either by Thiamet-G or O-GlcNAcase knockdown, promoted calcification of primary mouse vascular smooth muscle cells. Increased O-GlcNAcylation in diabetic arteries or in the O-GlcNAcase knockdown vascular smooth muscle cell upregulated expression of the osteogenic transcription factor Runx2 and enhanced activation of AKT. O-GlcNAcylation of AKT at two new sites, T430 and T479, promoted AKT phosphorylation, which in turn enhanced vascular smooth muscle cell calcification. Site-directed mutation of AKT at T430 and T479 decreased O-GlcNAcylation, inhibited phosphorylation of AKT at S473 and binding of mammalian target of rapamycin complex 2 to AKT, and subsequently blocked Runx2 transactivity and vascular smooth muscle cell calcification. Conclusions: O-GlcNAcylation of AKT at 2 new sites enhanced AKT phosphorylation and activation, thus promoting vascular calcification. Our studies have identified a novel causative effect of O-GlcNAcylation in regulating vascular calcification in diabetes mellitus and uncovered a key molecular mechanism underlying O-GlcNAcylation–mediated activation of AKT.

Activation of AKT by O-Linked N-Acetylglucosamine Induces Vascular Calcification in Diabetes MellitusNovelty and Significance

Rationale: Vascular calcification is a serious cardiovascular complication that contributes to the increased morbidity and mortality of patients with diabetes mellitus. Hyperglycemia, a hallmark of diabetes mellitus, is associated with increased vascular calcification and increased modification of proteins by O-linked N-acetylglucosamine (O-GlcNAcylation). Objective: We sought to determine the role of protein O-GlcNAcylation in regulating vascular calcification and the underlying mechanisms. Methods and Results: Low-dose streptozotocin-induced diabetic mice exhibited increased aortic O-GlcNAcylation and vascular calcification, which was also associated with impaired aortic compliance in mice. Elevation of O-GlcNAcylation by administration of Thiamet-G, a potent inhibitor for O-GlcNAcase that removes O-GlcNAcylation, further accelerated vascular calcification and worsened aortic compliance of diabetic mice in vivo. Increased O-GlcNAcylation, either by Thiamet-G or O-GlcNAcase knockdown, promoted calcification of primary mouse vascular smooth muscle cells. Increased O-GlcNAcylation in diabetic arteries or in the O-GlcNAcase knockdown vascular smooth muscle cell upregulated expression of the osteogenic transcription factor Runx2 and enhanced activation of AKT. O-GlcNAcylation of AKT at two new sites, T430 and T479, promoted AKT phosphorylation, which in turn enhanced vascular smooth muscle cell calcification. Site-directed mutation of AKT at T430 and T479 decreased O-GlcNAcylation, inhibited phosphorylation of AKT at S473 and binding of mammalian target of rapamycin complex 2 to AKT, and subsequently blocked Runx2 transactivity and vascular smooth muscle cell calcification. Conclusions: O-GlcNAcylation of AKT at 2 new sites enhanced AKT phosphorylation and activation, thus promoting vascular calcification. Our studies have identified a novel causative effect of O-GlcNAcylation in regulating vascular calcification in diabetes mellitus and uncovered a key molecular mechanism underlying O-GlcNAcylation–mediated activation of AKT.