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Approximately 20% of the world’s population will be around or above 65 years of age by the next decade. Out of these, 40% are suspected to have cardiovascular diseases as a cause of mortality. Arteriosclerosis, characterized by increased vascular calcification, impairing Windkessel effect and tissue perfusion, and determining end-organ damage, is a hallmark of vascular pathology in the elderly population. Risk factors accumulated during aging affect the normal physiological and vascular aging process, which contributes to the progression of arteriosclerosis. Traditional risk factors, age-associated diseases, and respective regulating mechanisms influencing vascular calcification and vascular stiffness have been extensively studied for many years. Despite the well-known fact that aging alone can induce vascular damage, specific mechanisms that implicate physiological aging in vascular calcification, contributing to vascular stiffness, are poorly understood. This review focuses on mechanisms activated during normal aging, for example, cellular senescence, autophagy, extracellular vesicles secretion, and oxidative stress, along with the convergence of premature aging models’ pathophysiology, such as Hutchinson-Gilford Progeria (prelamin accumulation) and Klotho deficiency, to understand vascular calcification in aging. Understanding the mechanisms of vascular damage in aging that intersect with age-associated diseases and risk factors is crucial to foster innovative therapeutic targets to mitigate cardiovascular disease. Visual Overview— An online visual overview is available for this article.

Objective— We hypothesized that ob/ob mice develop expansive vascular remodeling associated with calcification. Approach and Results— We quantified and investigated mechanisms of vascular remodeling and vascular calcification in ob/ob mice after vitamin D 3 (VD) stimulation or PBS (control), compared with C57BL/6 mice. Both ob/ob (OBVD [VD-treated ob/ob mice]) and C57BL/6 (C57VD [VD-treated C57BL/6 mice]) received 8×10 3 IU/day of intraperitoneal VD for 14 days. Control ob/ob (OBCT [PBS-treated ob/ob mice]) and C57BL/6 (C57CT [PBS-treated C57BL/6 mice]) received intraperitoneal PBS for 14 days. Hypervitaminosis D increased the external and internal elastic length in aortae from OBVD, resulting in increased total vascular area and lumen vascular area, respectively, which characterizes expansive vascular remodeling. OBVD decreased the aortic wall thickness, resulting in hypotrophic vascular remodeling. We demonstrated increased collagen deposition, elastolysis, and calcification in aortae from OBVD. Our results showed a positive correlation between expansive vascular remodeling and vascular calcification in OBVD. We demonstrated increased serum calcium levels, augmented Bmp (bone morphogenetic protein)-2 and osteochondrogenic proteins expression in OBVD aortae. Furthermore, aortae from OBVD increased oxidative stress, coincidently with augmented in situ MMP (matrix metalloproteinase) activity and exhibited no VDR (VD receptor) inhibition after VD. Conclusions— Our data provide evidence that obese and insulin-resistant mice (ob/ob) developed expansive hypotrophic vascular remodeling correlated directly with increased vascular calcification after chronic VD stimulation. Positive hypotrophic vascular remodeling and vascular calcification in this mouse model is possibly mediated by the convergence of absence VDR downregulation after VD stimulation, increased reactive oxygen species generation, and MMP activation.

Diabetes mellitus accelerates vascular calcification (VC) and increases the risk of end-stage renal disease (ESRD). Nevertheless, the impact of VC in renal disease progression in type 2 diabetes mellitus (T2DM) is poorly understood. We addressed the effect of VC and mechanisms involved in renal dysfunction in a murine model of insulin resistance and obesity (ob/ob), comparing with their healthy littermates (C57BL/6). We analyzed VC and renal function in both mouse strains after challenging them with Vitamin D3 (VitD3). Although VitD3 similarly increased serum calcium and induced bone disease in both strains, 24-hour urine volume and creatinine pronouncedly decreased only in ob/ob mice. Moreover, ob/ob increased urinary albumin/creatinine ratio (ACR), indicating kidney dysfunction. In parallel, ob/ob developed extensive intrarenal VC after VitD3. Coincidently with increased intrarenal vascular mineralization, our results demonstrated that Bone Morphogenetic Protein-2 (BMP-2) was highly expressed in these arteries exclusively in ob/ob. These data depict a greater susceptibility of ob/ob mice to develop renal disease after VitD3 in comparison to paired C57BL/6. In conclusion, this study unfolds novel mechanisms of progressive renal dysfunction in diabetes mellitus (DM) after VitD3in vivo associated with increased intrarenal VC and highlights possible harmful effects of long-term supplementation of VitD3 in this population.

Background Coronary artery calcification (CAC) and atherosclerotic inflammation associate with increased risk of myocardial infarction (MI). Vascular calcification is regulated by osteogenic proteins (OPs). It is unknown whether an association exists between CAC and plasma OPs and if they are affected by atherothrombotic inflammation. We tested the association of osteogenic and inflammatory proteins with CAC and assessed these biomarkers after MI. Methods Circulating OPs (osteoprotegerin, RANKL, fetuin-A, Matrix Gla protein [MGP]) and inflammatory proteins (C-reactive protein, oxidized-LDL, tumoral necrosis factor-α, transforming growth factor [TGF]-β1) were compared between stable patients with CAC (CAC ≥ 100 AU, n = 100) and controls (CAC = 0 AU, n = 30). The association between biomarkers and CAC was tested by multivariate analysis. In patients with MI (n = 40), biomarkers were compared between acute phase and 1-2 months post-MI, using controls as a baseline. Results MGP and fetuin-A levels were higher within individuals with CAC. Higher levels of MGP and RANKL were associated with CAC (OR 3.12 [95% CI 1.20-8.11], p = 0.02; and OR 1.75 [95% CI 1.04-2.94] respectively, p = 0.035). After MI, C-reactive protein, OPG and oxidized-LDL levels increased in the acute phase, whereas MGP and TGF-β1 increased 1-2 months post-MI. Conclusions Higher MGP and RANKL levels associate with CAC. These findings highlight the potential role of these proteins as modulators and markers of CAC. In addition, the post-MI increase in OPG and MGP, as well as of inflammatory proteins suggest that the regulation of these OPs is affected by atherothrombotic inflammation.

Vascular calcification (VC) is an active pathophysiological process promoting enhanced morbidity and mortality without effective therapy. Oxidative stress and endoplasmic reticulum (ER) stress support cardiovascular calcification progression. We showed previously that the ER redox chaperone protein disulfide isomerase-A1 (PDI) is required for agonist-driven Nox NADPH oxidase activation. Oxidant levels and expression of Nox subunits and PDI are upregulated in calcifying rabbit valves. We hypothesized that PDI supports VC. We recently developed a new PDI overexpression mouse model (TgPDI) in FVB background (WT). To investigate whether TgPDI mice show increased VC, we challenged 20 week-old TgPDI mice with Vitamin-D 3 (VitD) for 14 days. Also, we incubated primary vascular smooth muscle cells (VSMC) isolated from WT or TgPDI mice with mineralizing medium (β-glycerophosphate 10mM + CaCl 2 6mM). TgPDI results were compared to those from respective WT littermates. We tested distinct VitD doses. At 9x10 4 IU/day, WT mice depicted negligible increases in calcification area (median 0.07% to 0.36% aorta cross-sectional area, p=ns, n=5), while TgPDI showed enhanced calcification (median 0.20 to 3.63 % area, pIn vitro, VSMC phenotype marker expression (SM22 and alpha-actin) was enhanced in TgPDI vs. WT cells at baseline, while calcifying stimulation for 3 or 7 days decreased such VSMC marker expression both in WT and in TgPDI VSMC. Conversely, calcifying stimulation for 3 or 7 days showed no effects on ER stress marker expression (GRP78, GRP94). To assess whether these findings apply to humans, we examined femoral arteries from diabetic and non-diabetic patients with peripheral arterial disease, which showed increased arterial calcification and marked coincident PDI overexpression in samples from diabetic patients. In conclusion, PDI overexpression in vivo associates with enhanced VC after VitD calcifying stimulus. PDI and its associated signaling proteins deserve further study as potential therapeutic targets in VC progression.

Osteogenic Proteins (OP) and atherosclerotic inflammation modulate vascular calcification. Patients with acute myocardial infarction (MI) demonstrate higher inflammation and develop accelerated coronary artery calcification (CAC), compared to stable coronary artery disease patients. We aimed to: (a) test the association of OP and inflammatory proteins with CAC in individuals assessed for primary cardiovascular prevention; (b) evaluate these biomarkers in acute and subacute phases post-MI. We prospectively enrolled 170 patients, divided in 3 groups: (1) primary prevention patients who underwent ambulatory coronary computed tomography with a CAC score ≥ 100, n=100; (2) primary prevention patients with a CAC score=zero, n=30; (3) post-MI patients, n=40, during acute (3±1 days post-MI) and subacute (46±17 days post-MI) phases. Serum OP (osteoprotegerin, RANKL, fetuin-A and Matrix Gla protein [MGP]) and serum inflammatory proteins (C-reactive protein, oxidized LDL, tumoral necrosis factor-α and tumor growth factor [TGF]-β1) were measured by ELISA. Associations of plasma OP and inflammatory proteins levels with CAC were adjusted by age, sex and use of statins. In the post-MI group, all biomarkers were compared both in acute vs. subacute phases and in acute phase vs. CAC zero group. Compared to CAC zero, patients with CAC score ≥ 100 were older, predominantly men and had higher rates of hypertension and diabetes. After adjusted analysis, only MGP was associated with a CAC score ≥ 100 (OR 1.48 [95% CI 1.01-2.16; per 100ng/ml increase; p=0.047). Serum MGP and TGF-β1 were higher in subacute phase compared to acute phase post-MI (median [25-75 percentile], 342 vs. 279ng/ml respectively; p=0.01 and 711 vs. 492pg/ml; p=0.03). All biomarkers were similar in acute phase post-MI vs. CAC zero. In conclusion, serum MGP was associated with higher CAC in primary cardiovascular prevention patients. After MI, both serum MGP and TGF-β1 levels increased in the subacute phase. Our findings unveiled a potential role for MGP as a biomarker of CAC in primary cardiovascular prevention. Additionally, this clinical study unfolds further mechanistic hypothesis of how increased TGF-β1 and MGP may modulate vascular inflammation and CAC progression in subacute MI patients.

Over the past decade, we have witnessed an explosion of fundamental research aimed at understanding mechanisms contributing to cardiovascular calcification. As highlighted in recent reviews, numerous animal models and patient group studies have lent key insights into mechanisms and processes underlying pathological remodeling of soft tissues,1 including activation of signaling cascades related to bone morphogenetic proteins,2 Wnt/β-catenin,3,4 matrix γ-carboxyglutamate (Gla) protein (MGP),5,6 transforming growth factor (TGF)-β, phosphate signaling,7,8 and various downstream targets. Although there are compelling data supporting the biological importance of these pathways, harnessing these mechanisms for the development of therapeutics has not yet been realized. Many pathways play an integral role in bone homeostasis, making systemic targeting a nonviable therapeutic approach. In this Recent Highlights focused on cardiovascular calcification, we have drawn from the pool of recent publications in ATVB and other leading journals that focus on genetic and nongenetic upstream modulators of ectopic calcification pathways, and we posit that interventions aimed at reducing their impact may be more readily translated to clinical therapies for patients. A greater understanding of the key local and systemic cofactors, initiators, and outcomes will create a complementary approach to advancing both science and medicine. We further argue that identification of biomarkers that are prognostic not only for the presence of vascular calcification (VC) but also for the rate of progression of VC will be instrumental in the early identification and appropriate management of patients in the future. Unlike metastatic calcification—caused by elevated levels of calcium in the blood—cardiovascular calcification is most often attributed to injury or maladaptive cellular responses to stress. Although in vitro studies and genetically modified model organisms can serve as useful platforms to understand the biology of disease phenotypes, the reality is that >85% of drugs stemming from …

Background and aims Obesity and diabetes potentiate vascular calcification by increasing vascular smooth muscle cells osteoblastic differentiation mediated by the transcription factor Msx2 and bone morphogenetic protein-2 signaling. However, Bmp-2/Msx2 crosstalk to induce VSMC osteogenic phenotype transition and calcification is poorly understood in diabetes. We aimed to investigate mechanisms underlying Bmp-2-driven VSMC osteogenic differentiation and calcification in leptin-deficient ob/ob mice. Methods We incubated VSMC from ob/ob mice and wild type C57BL/6 littermates with or without Bmp-2. We used loss-of-function experiments to investigate the role of Msx2 in Bmp-2-induced ob/ob VSMC osteochondrogenic differentiation and calcification by transfecting Msx2 siRNA into VSMC. Results Baseline ob/ob VSMC and aorta showed increased Msx2, Runx2, alkaline phosphatase mRNA and protein expression, which further increased in Bmp-2-incubated ob/ob VSMC, therefore augmenting ob/ob VSMC calcification in comparison to wild type VSMC. Accordingly, signaling pathways to induce VSMC osteogenic differentiation, such as Smad1/5 phosphorylation increased in ob/ob versus wild type aorta. In comparison to wild type VSMC, Msx2 siRNA transfected VSMC decreased Bmp-2-dependent osteochondrogenic differentiation response by abrogating Msx2, Runx2, Alpl expression in ob/ob but not in wild type VSMC. Nonetheless, Msx2 inhibition did not decrease calcification in Bmp-2 stimulated ob/ob VSMC in vitro. Conclusions Our data support a crucial role of Msx2 for ob/ob VSMC osteochondrogenic differentiation, however, Msx2 signaling alone is not sufficient for ob/ob VSMC calcification after Bmp-2 stimulation in vitro. These findings can be translated into novel perspectives for the understanding of the mechanisms and to provide therapeutic targets underlying vascular calcification in type 2 diabetes.

A calcificação vascular na doença arterial coronária está ganhando importância, tanto em pesquisas científicas como em aplicações clínicas e de imagem. A placa calcificada é considerada a forma mais relevante de aterosclerose dentro da árvore arterial coronária e frequentemente apresenta um desafio para a intervenção percutânea. Estudos recentes têm demonstrado que a calcificação da placa é dinâmica e está estreitamente ligada ao grau de inflamação vascular. Vários fatores inflamatórios, produzidos durante as diferentes fases da aterosclerose, induzem a expressão e ativação de células osteoblásticas localizadas na parede arterial, que, por sua vez, promovem a deposição de cálcio. As células do músculo liso vascular possuem uma capacidade extraordinária de sofrer diferenciação fenotípica osteoblástica. Não há dúvida de que o papel desses fatores, bem como os elementos de genômica e proteômica, poderia ser um ponto estratégico fundamental na prevenção e no tratamento. Neste contexto, realizamos uma atualização sobre a calcificação coronária, com foco em fisiopatologia, modelos experimentais e implicações clínicas da calcificação vascular. Vascular calcification in coronary artery disease is gaining importance, both in scientific research and in clinical and imaging applications. The calcified plaque is considered the most relevant form of atherosclerosis within the coronary artery tree and is frequently a challenge for percutaneous intervention. Recent studies showed that plaque calcification is dynamic and is strictly related to the degree of vascular inflammation. Several inflammatory factors produced during the different phases of atherosclerosis induce the expression and activation of osteoblastic cells located within the arterial wall, which, in turn, promote the deposit of calcium. The vascular smooth muscle cells have an extraordinary capacity to undergo osteoblastic phenotypical differentiation. There is no doubt that the role of these factors, as well as the elements of genomics and proteomics, could be a vital strategic point in prevention and treatment. Within this context, we conducted an updating review on coronary calcification focused on pathophysiology, experimental models, and clinical implications of vascular calcification.

Vascular calcification is a tightly regulated process that increases during ageing and occurs mainly in patients with diabetes and chronic renal failure. Exosomes are small membrane vesicles that are synthesized in a particular population of endosomes, also called multivesicular bodies, by inside budding into the lumen of the compartment. After fusion of exosome with the plasma membrane, these internal vesicles are secreted. Exosomes have a defined set of membrane and cytosolic proteins. The physiological function of exosomes is still a matter of debate. Investigators implicated microvesicles/exosomes as a specific signaling mechanism to induce vascular mineralization during vascular smooth muscle cells phenotypic transition. Vascular wall from healthy individual exhibit exosomes loaded with calcification inhibitors such as Fetuin A and MGP. Conversely, calcifying conditions induce secretion of exosomes, characterized by decreased calcifying inhibitors and increased phosphatidyl serine and Annexin A6 content, which serves as a nidus for vascular calcification.

Vascular remodeling, which is an adaptive response of the vessel to specific stimuli has a pivotal role in cardiovascular disease, such as aneurysm formation. In that scope, serum endothelin-1 (ET-1) which is increased in diabetic patients, may contribute to atherosclerosis and aneurysm development. We hypothesized that leptin-deficient ob/ob mice may demonstrate an exaggerated vascular remodeling associated with vascular calcification after Vitamin D 3 in vivo . We further investigated the role of ET-1 in vascular smooth muscle cells (SMC) osteochondrogenic differentiation and calcification in vitro . We used leptin-deficient (obob, n=12) and C57BL/6 mice (C57, n=12) injected with saline (Cont) or Vitamin D 3 8x10 4 IU (VitD) i.p. daily for 14 days. Results are shown as Mean±SEM and considered statistically significant if p≤.05 by ANOVA. Aorta from ob/obVitD increased both external and internal elastic lamina circumferential length (3478±269.7μm and 3186±262.6μm) versus (vs.) ob/obCont (2841±74.73μm and 2657±71.37μm) vs. C57VitD (2611±39.62μm and 2333±41.79μm) and vs. C57Cont (2569±164.6μm and 2351±167.7μm), thus characterizing outward vascular remodeling. We found that vascular calcification area strongly correlated with increased total vessel area (r 2 =0.8, p=0.003). We also incubated primary SMC isolated either from obob or from C57 aorta without (Cont) or with ET-1 50nM (ET) for 0-72h. ObobET SMC increased calcification vs. obobCont (1,69±0.025 vs. 1.03±0.03) after 14 days pin vivo . Nevertheless, C57VitD increased ETA receptor mRNA expression (5.44±1.08) only. In conclusion, we uncovered signaling pathways that may connect Vitamin D and ET-1 calcifying effect to induce excessive outward remodeling in type 2 diabetes.

Objective— We hypothesized that reactive oxygen species (ROS) contribute to progression of aortic valve (AV) calcification/stenosis. Methods and Results— We investigated ROS production and effects of antioxidants tempol and lipoic acid (LA) in calcification progression in rabbits given 0.5% cholesterol diet +10 4 IU/d Vit.D 2 for 12 weeks. Superoxide and H 2 O 2 microfluorotopography and 3-nitrotyrosine immunoreactivity showed increased signals not only in macrophages but preferentially around calcifying foci, in cells expressing osteoblast/osteoclast, but not macrophage markers. Such cells also showed increased expression of NAD(P)H oxidase subunits Nox2, p22phox, and protein disulfide isomerase. Nox4, but not Nox1 mRNA, was increased. Tempol augmented whereas LA decreased H 2 O 2 signals. Importantly, AV calcification, assessed by echocardiography and histomorphometry, decreased 43% to 70% with LA, but increased with tempol ( P ≤0.05). Tempol further enhanced apoptosis and Nox4 expression. In human sclerotic or stenotic AV, we found analogous increases in ROS production and NAD(P)H oxidase expression around calcifying foci. An in vitro vascular smooth muscle cell (VSMC) calcification model also exhibited increased, catalase-inhibitable, calcium deposit with tempol, but not with LA. Conclusions— Our data provide evidence that ROS, particularly hydrogen peroxide, potentiate AV calcification progression. However, tempol exhibited a paradoxical effect, exacerbating AV/vascular calcification, likely because of its induced increase in peroxide generation.

β-actin, a highly conserved gene/protein, which is usually not modulated by different stimuli, is routinely used to normalize target gene or protein expression in experimental biology, and so defined endogenous housekeeping gene. However, investigators showed that β-actin expression could change during cellular growth and differentiation. We hypothesized that endothelin 1 (ET-1), a cellular proliferative agonist, increases β-actin expression and stimulates calcification in vascular smooth muscle cells (SMC) from obob mice. Primary aortic SMC passage 4-6 isolated from obese leptin-deficient (obob) and from C57BL/6 (C57) mice were incubated without (Cont) or with ET-1 50nM (ET) for 0-72h. Cell lysates were assessed for β-actin and RUNX2 expression by western blotting and normalized by GAPDH. ObobET SMC increased β-actin expression after 24h, 48h and 72h (1.68±0.06, 1.66±0.05, 1.73±0.06 respectively) vs obobCont (1.01±0.07), p

Vascular calcification decreases compliance and increases morbidity. Mechanisms of this process are unclear. The role of oxidative stress and effects of antioxidants have been poorly explored. We investigated effects of the antioxidants lipoic acid (LA) and tempol in a model of atherosclerosis associated with elastocalcinosis. Male New Zealand white rabbits (2.5-3.0 kg) were fed regular chow (controls) or a 0.5% cholesterol (chol) diet+104 IU/day vitamin D2 (vitD) for 12 weeks, and assigned to treatment with water (vehicle, n=20), 0.12 mmol·kg-1·day-1 LA (n=11) or 0.1 mmol·kg-1·day-1 tempol (n=15). Chol+vitD-fed rabbits developed atherosclerotic plaques associated with expansive remodeling, elastic fiber disruption, medial calcification, and increased aortic stiffness. Histologically, LA prevented medial calcification by ∼60% and aortic stiffening by ∼60%. LA also preserved responsiveness to constrictor agents, while intima-media thickening was increased. In contrast to LA, tempol was associated with increased plaque collagen content, medial calcification and aortic stiffness, and produced differential changes in vasoactive responses in the chol+vitD group. Both LA and tempol prevented superoxide signals with chol+vitD. However, only LA prevented hydrogen peroxide-related signals with chol+vitD, while tempol enhanced them. These data suggest that LA, opposite to tempol, can minimize calcification and compliance loss in elastocalcionosis by inhibition of hydrogen peroxide generation.

Vascular calcification is a regulated process, which associates with coronary artery disease (CAD) and occurs through an increase in transcription factor expression such as RUNX2, MSX2 and alkaline phosphatase (ALP), then inducing calcium deposition. Bone morphogenetic protein-2 (BMP2) is a potent osteochondrogenic mediator, which is expressed in CAD. Endothelin-1 (ET1) and leptin have a role in regulating inflammation and CAD. We hypothesized that BMP2, leptin or both increase ROS formation in C57BL/6 vascular smooth muscle cells (SMC), stimulating osteochondrogenic differentiation. We also investigated the effect of ET1 in SMC osteochondrogenesis. Our objectives were: to investigate ROS production in SMC after BMP2 (50 ng/ml) and/or leptin (10 ng/ml) incubation for 6 hours; and to assess osteochondrogenic gene expression and calcification of SMC stimulated with BMP2, leptin or ET1 (10 nM).

Accumulating evidence indicates that vascular dysfunction in atherosclerosis, hypertension, and diabetes is either caused by or accompanied by oxidative stress in the vessel wall. In particular, the role of redox processes as mediators of vascular repair and contributors to post-angioplasty restenosis is increasingly evident. Yet the pathophysiology of such complex phenomena is still unclear. After vascular injury, activation of enzymes such as NADPH oxidase leads to a marked increase in superoxide generation, proportional to the degree of injury, which rapidly subsides. Such early superoxide production is significantly greater after stent deployment, as compared to balloon injury. Recent data suggest the persistence of low levels of oxidant stress during the vascular repair reaction in neointimal and medial layers. Despite the compensatory increase in expression of iNOS and nNOS, nitric oxide bioavailability is reduced because of increased reaction rates with superoxide, yielding as by-products reactive nitrogen/oxygen species that induce protein nitration. Concurrently, the activity of vascular superoxide dismutases exhibits a sustained decrease following injury. This decreased activity appears to be a key contributor to vasoconstrictive remodeling and a major determinant of the occurrence of nitrative/oxidative stress. Replenishment of superoxide dismutase (SOD), as well as treatment with vitamins C and E or the lipid-lowering drug probucol and its analogs, led to decrease in constrictive remodeling and improved vessel caliber. Better understanding of the redox pathophysiology of vascular repair should help clarify the pathogenesis of many other vascular conditions and may provide novel therapeutic strategies to prevent vascular lumen loss.