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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.

Vascular calcification associates with increased cardiovascular morbidity and mortality in patients with diabetes mellitus and end-stage renal disease. We evaluated intrarenal vascular calcification and renal dysfunction after either vitamin D 3 (VitD 3 4.4-6.4x104 IU/day i.p. for 21 days) or saline in ob/ob mice (n=12), a murine model of type 2 diabetes mellitus (T2DM), in comparison to paired C57BL/6 mice (n=12). The animal protocol #2242-14 was approved by Institutional ethics committee. VitD3 increased serum calcium levels in both animal strains. Serum creatinine increased in VitD3-treated mice (92% and 43% in ob/ob and C57BL/6 respectively), while serum urea levels decreased in ob/ob and increased in C57BL/6 after calcifying protocol. Both VitD 3 -treated ob/ob and C57BL/6 decreased 24-hour urine volume, which was more pronounced in diabetic animals (~5 times and ~1.5 reduction in ob/ob and in C57BL/6 respectively). Moreover, creatinine clearance decreased in VitD 3 -treated mice, especially in ob/ob animals (~7 and ~3 fold reduction in ob/ob and C57BL/6, respectively). Additionally, urinary albumin/creatinine ratio increased after VitD 3 administration in ob/ob mice only (~13 times in comparison to control ob/ob). Alizarin Red S, Von Kossa and Osteosense TM revealed that only ob/ob mice developed extensive intrarenal vascular calcification after VitD 3 . Interestingly, histomorphometric analysis of the femur showed that VitD 3 induced similar osteomalacia in both ob/ob and C57BL/6 mice. Coincidently with increased intrarenal vascular mineralization in ob/ob, confocal immunofluorescence analysis demonstrated that Bone Morphogenetic Protein-2 (BMP-2) was highly expressed in these arteries exclusively in ob/ob. These data suggest a greater susceptibility of T2DM murine model to VitD 3 , in comparison to paired C57BL/6 littermates to induce kidney dysfunction associated with increased intrarenal vascular calcification, together with BMP-2 signaling pathway activation. These findings were independent of bone remodeling and serum calcium levels. In conclusion, this study unfolded novel mechanisms of progressive renal dysfunction in T2DM after VitD 3 in vivo associated with increased intrarenal vascular calcification.

Introduction: Diabetes mellitus (DM) accelerates vascular calcification (VC) in peripheral arterial disease (PAD), increasing limb ischemia and amputation risk. Although DM and VC implicates in PAD, the mechanisms underlying vascular smooth muscle cells (SMC) osteochondrogenic differentiation in this setting are scarce. Objectives: To assess VC and osteogenic mRNA/protein expression (Msx2, Runx2 and ALPL) in arteries and in primary SMC isolated from PAD patients with DM (n=3), without DM (n=2) and individuals without PAD (control [CTL], n=2) that underwent amputation. Methods: Ethical committee approved study and patients signed informed consent. 5-7 th passages SMC were incubated without (untreated) or with β-glycerophosphate 10mM (β-GP) for 48h (mRNA expression), 72h (protein expression) and 14 days (calcification). In addition, we performed tissue immunofluorescence and Alizarin Red S. Results: Untreated PAD+DM SMC increased calcification (4.6±1.2) in comparison to PAD without DM patients (2.5±0.2) and CTL individuals (1.0±0.07). β-GP further increased calcification in respective groups (51.3±4.7; 13.5±1.1 and 9.8±1.2). Msx2 mRNA expression decreased in CTL SMC after β-GP (48h) and did not change in PAD without DM and in PAD+DM SMC. Msx2 protein expression decreased in all groups after calcifying medium (72h). Moreover, Runx2 mRNA expression increased in β-GP-treated PAD+DM SMC and ALPL mRNA expression augmented in β-GP-treated SMC from PAD without DM patients. However, Runx2 and ALPL protein expression was not modulated by calcifying medium (72h) in all groups. Interestingly, immunofluorescence of arterial samples demonstrated increased osteochondrogenic protein expression (Msx2, Runx2 and ALPL) around calcifying foci in PAD+DM group (+++) versus PAD without DM (++) and versus CTL (+) patients. Coincidently, PAD+DM group showed augmented vascular calcification (19x10 4 ±10x10 4 ; 7x10 4 ±2x10 4 ; 3x10 3 ±10 -2 μm 2 ). Conclusion: We demonstrated increased calcification in arteries from amputated PAD+DM patients and in respective SMC without and with calcifying medium. Although we found augmented osteochondrogenic protein expression in the vessel wall from PAD+DM individuals this was not robustly shown in isolated SMC in vitro .

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.

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.

Vascular calcification may determine arterial flow imbalance through increasing vascular rigidity and Windkessel effect, frequently occurring in diabetes. Vascular remodeling, considered an adaptive response of the vessel to specific stimuli, may associate with calcification and participate in pathophysiology of cardiovascular disease. In order to further investigate the intersection between pathophysiology of vascular calcification and vascular remodeling, we hypothesized that leptin-deficient ob/ob mice, demonstrate increased vascular remodeling and vascular calcifying response to Vitamin D 3 in vivo compared to C57BL/6 (C57) mice. We injected C57 and ob/ob mice with Vitamin D 3 8x10 4 IU (VitD) daily or saline 0.9% ip. (Cont) for 14 days and for 21 days. Results are showed as Mean±SEM. We considered statistically significant if pb/ob VitD aorta decreased aortic wall thickness (24.3±0.4μm) versus ob/ob Cont (29.7±1.3μm), vs. C57VitD (34.4±0.6μm) and vs. C57Cont (39±1.3μm), pob/ob VitD decreased aortic wall thickness (25.4±0.5μm) vs. ob/ob Cont (29.7±1.3μm) and vs. C57Cont (39.0±1.3μm), pb/ob VitD decreased vessel wall area (VWA)= 79120±3957μm 2 vs. ob/ob Cont=85310±4593μm 2 and vs. C57Cont=96970±6719μm 2 after 14d (n=6) and also after 21d ( ob/ob VitD=72090±3268μm 2 vs. ob/ob Cont=85310±4593μm 2 (n=3). On contrary, C57VitD did not change VWA. Coincidently, ob/ob VitD increased vascular calcification after 14d (total Ca ++ area burden=4548.2μm 2 , n=6) and after 21d (total Ca ++ area burden=2212.88 μm 2 , n=3) respectively. C57VitD and C57Cont did not calcify (n=6). Together with increased VWA loss and calcification, ob/ob VitD also showed augmented elastolysis and fibrosis. Nevertheless, hypotrophic remodeling did not determine any change in vascular lumen area ( ob/ob VitD=208300±24810μm 2 ; ob/ob Cont=207300±22740μm 2 , C57VitD=196800±22120μm 2 and C57Cont=225400±17960μm 2 ) after 14d in this model. Vascular remodeling associated with calcification and other architectural changes of the vascular wall may bring important insights for investigating human cardiovascular disease in obese and insulin resistant background.

Peripheral artery disease (PAD) frequently occurs in diabetes mellitus (DM) and associates with limb ischemia and amputation. Vascular calcification, which is very prevalent both in DM and in the elderly, contributes to PAD pathophysiology and further increases amputation risk. Our objectives were to quantify vascular calcification, elastic and collagen fiber content, besides osteochondrogenic protein expression in arteries from 2 diabetic patients at the extremes of age. Femoral and dorsalis pedis artery from a 92-year-old male (O) and a 40-year-old male (Y), both with DM, were harvested at the time of amputation caused by limb ischemia. Other cardiovascular risk factors were similar among patients. Specimens were fixed in buffered formaldehyde, processed and stained for hematoxylin-eosin, Verhoeff-van-Gieson and Masson’s trichrome. Immunohistochemical analysis was performed to assess osteogenic protein expression such as RUNX2, alkaline phosphatase (ALP) and Osterix (OSX), which are upregulated during vascular smooth muscle cell reprogramming to an osteogenic phenotype, responsible for hydroxyapatite deposition. Arteries from 92-year-old patient showed markedly architectural changes of the vascular wall in comparison with the younger patient sample, such as decreased collagen (O=0.8±0.2% versus Y=33.5±5.2% area/field, p2 /field vs. Y=30.6±6.3 mm 2 /field). Osteochondrogenic protein expression also showed opposing results: OSX stained around large calcification nuclei, mostly in the older, but not in the younger patient samples. Furthermore, RUNX2 and ALP expression, which determines early osteoblastic cell differentiation, occurred around small calcium deposits, mainly in the 40-year-old patient. We demonstrated that age and time-length exposure to diabetes are predictive of collagen and elastic fibers content associated with increased calcification area. Moreover, osteochondrogenic protein expression profile favors RUNX2 and ALP in the younger vs OSX in the older patient respectively, implying different stages of the disease.

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