Scaffold-free human vascular calcification model using a bio-three-dimensional printer.

Morbidity and mortality rates associated with atherosclerosis-related diseases are increasing. Therefore, developing new research models is important in furthering our understanding of atherosclerosis and investigate novel treatments. Here, we designed novel vascular-like tubular tissues from multicellular spheroids composed of human aortic smooth muscle cells, endothelial cells, and fibroblasts using a bio-3D printer. We also evaluated their potential as a research model for Mönckeberg's medial calcific sclerosis. The tubular tissues were sufficiently strong to be handled 1 week after printing and could still be cultured for 3 weeks. Histological assessment showed that calcified areas appeared in the tubular tissues within 1 week after culture in a medium containing inorganic phosphate (Pi) or calcium chloride as the calcification-stimulating factors. Calcium deposition was confirmed using micro-computed tomography imaging. Real-time quantitative reverse transcription polymerase chain reaction analysis revealed that the expression of osteogenic transcription factors increased in calcified tubular tissues. Furthermore, the administration of Pi and rosuvastatin enhanced tissue calcification. The bio-3D printed vascular-like tubular structures, which are composed of human-derived cells, can serve as a novel research model for Mönckeberg's medial calcific sclerosis.

Mechanisms of Arterial Calcification: The Role of Matrix Vesicles.

Vascular calcification is related to vascular diseases, for example, atherosclerosis, and its comorbidities, such as diabetes and chronic kidney disease. In each condition, a distinctive histological pattern can be recognised that may influence technical choices, possible intra-operative complications, and procedure outcomes, no matter if the intervention is performed by open or endovascular means. This review considers the classification and initiating mechanisms of vascular calcification. Dystrophic and metastatic calcifications, Monckeberg's calcification, and genetic forms are firstly outlined, followed by their alleged initiation mechanisms; these include (a) ineffective macrophage efferocytosis; (b) ectopic osteogenesis driven by modified resident or circulating osteoprogenitors. As in physiological bio-mineralisation, active calcification starts with the deposition of cell derived matrix vesicles into the extracellular matrix. To substantiate this belief, an in depth ultra-structural documentation of hydroxyapatite crystal deposition on such vesicles is provided in an ex-vivo human vascular cell model. Revealing the vesicle composition and phenotype in normal and pathological vascular conditions will be essential for the development of new therapeutic strategies, in order to prevent and treat vascular calcification.

Roles of High Mobility Group Box 1 in Cardiovascular Calcification.

Calcific disease of the cardiovascular system, including atherosclerotic calcification, medial calcification in diabetes and calcific aortic valve disease, is an important risk factor for many adverse cardiovascular events such as ischemic cardiac events and subsequent mortality. Although cardiovascular calcification has long been considered to be a passive degenerative occurrence, it is now recognized as an active and highly regulated process that involves osteochondrogenic differentiation, apoptosis and extracellular vesicle release. Nonetheless, despite numerous studies on the pathogenesis of cardiovascular calcification, the underlying mechanisms remain poorly understood. High mobility group box 1 (HMGB1), a nuclear protein bound to chromatin in almost all eukaryotic cells, acts as a damage-associated molecular pattern (DAMP) when released into the extracellular space upon cell activation, injury or death. Moreover, HMGB1 also functions as a bone-active cytokine participating in bone remodeling and ectopic calcification pathogenesis. However, studies on the roles of HMGB1 in promoting cardiovascular calcification are limited to date, and the mechanisms involved are still unclear. In this review, we summarize recent studies investigating the mechanism of cardiovascular calcification and discuss multiple roles of HMGB1 in its development.

Medial Arterial Calcification: An Overlooked Player in Peripheral Arterial Disease.

Peripheral arterial disease (PAD) is a global health issue that is becoming more prevalent in an aging world population. Diabetes mellitus and chronic kidney disease are also on the increase, and both are associated with accelerated vascular calcification and an unfavorable prognosis in PAD. These data challenge the traditional athero-centric view of PAD, instead pointing toward a disease process complicated by medial arterial calcification. Like atherosclerosis, aging is a potent risk factor for medial arterial calcification, and accelerated vascular aging may underpin the devastating manifestations of PAD, particularly in patients prone to calcification. Consequently, this review will attempt to dissect the relationship between medial arterial calcification and atherosclerosis in PAD and identify common as well as novel risk factors that may contribute to and accelerate progression of PAD. In this context, we focus on the complex interplay between oxidative stress, DNA damage, and vascular aging, as well as the unexplored role of neuropathy.

[Mönckeberg's sclerosis -- crystal-induced angiopathy]. / Mönckeberg-sclerosis -- kristály indukálta angiopathia.

INTRODUCTION: Mönckeberg's sclerosis is a special form of arteriosclerosis characterized by calcification and ossification of the media of medium size arteries mainly of lower extremities. AIMS: The aim of the authors was to examine medium size arteries with Mönckeberg's sclerosis in 22 amputated lower legs of 16 patients in order to demonstrate different crystals in the wall of blood vessels. METHODS: The methodology was based on previous findings of the authors indicating that in different metabolic disorders many crystals remain demonstrable in unstained histological sections unlike in haematoxylin-eosin stained sections. RESULTS: In unstained sections the authors observed rhomboid or prismatic calcium pyrophosphate dihydrate and clusters of elongated narrow hydroxyapatite crystals in the wall of medium size arteries of all examined cases. Both types of crystals showed axis parallel positive birefringence under polarized light. The intensity of birefringence of hydroxyapatite crystals was weaker in comparison with that of calcium pyrophosphate dihydrate crystals. Occasionally, other crystals which were different in shape from both calcium pyrophosphate dihydrate and hydroxyapatite crystals were also observed. CONCLUSIONS: It seems likely that similarly to crystal deposition induced arthropathy, calcium pyrophosphate dihydrate, hydroxyapatite and other crystals cause fibrosis and intimal proliferation, which may contribute to progressive occlusion of blood vessels resulting in ischemic symptoms. Based on this observation Mönckeberg's sclerosis may be defined as a crystal-induced angiopathy.

Clinical detection, risk factors, and cardiovascular consequences of medial arterial calcification: a pattern of vascular injury associated with aberrant mineral metabolism.

Patients with end-stage renal disease are characterized by extensive vascular calcification and high cardiovascular disease (CVD) risk. Calcification in end-stage renal disease patients represents at least two distinct pathologic processes. Calcification within the tunica intima frequently is associated with lipid-laden, flow-limiting atherosclerotic plaques. These appear as spotty areas of calcification interspersed with noncalcified arterial segments on plain radiography and generally are found near arterial branch points in medium-sized conduit arteries. In contrast, medial arterial calcification (MAC) involves deeper layers of the arterial wall; tends to affect the artery diffusely, appearing as a linear contiguous tram-track pattern of calcification on plain radiography; and often involves smaller muscular arteries such as the radial artery, intermammary arteries, and arteries in the ankle and foot. Both are related to CVD events, but potentially through different mechanisms. Atherosclerotic calcification may be marking the total burden of atherosclerosis, whereas MAC may lead to arterial stiffness and left ventricular hypertrophy. Existing data suggest that altered mineral metabolism may promote MAC, whereas heightened inflammation and oxidative stress contribute to atherosclerosis. Dysregulation of normal anticalcification factors and elastin degradation are common to both processes. Risk of vascular calcification also may be increased by the use of certain medications in the setting of chronic kidney disease. This review compares and contrasts known risk factors for MAC and atherosclerosis, describes existing and emerging technologies to distinguish between them, and reviews the existing literature linking each with CVD events in dialysis patients and in other settings.

[Comparative characteristic of energy supply disturbances in arterial and venous walls of rabbits with alloxan diabetes and monoiodacetate intoxication].

We showed here that energy metabolism in thoracic rabbit aorta and posterior vena cava is disturbed two weeks following the induction of alloxan-induced diabetes and monoiodacetate intoxication. In the vessels studied, the alterations are manifested in the decrease in the intensity of glucose uptake, oxygen consumption, lactic acid production, and ATP resynthesis. Simultaneously, the ATP content is significantly reduced. The possible significance of these disorders in the development of Monckeberg's arteriosclerosis is discussed.

Accelerated atherosclerotic calcification and Monckeberg's sclerosis: a continuum of advanced vascular pathology in chronic kidney disease.

Autopsy studies have demonstrated the near universal presence of fatty streaks and fibroatheromas in the general population from which patients with chronic kidney disease (CKD) arise. The vast majority of patients with CKD have multiple conventional cardiovascular risk factors. Vascular atherosclerotic calcification develops in most patients as they transition from the general population to significant CKD as part of cholesterol crystallization within atherosclerotic lesions. Once present, however, atherosclerotic medial calcification can become prominent and has been previously identified as Mönckeberg's sclerosis. A unifying concept supported by the preponderance of pathologic evidence contends that Mönckeberg's sclerosis is a manifestation of accelerated atherosclerosis in patients with CKD. The term has also been used in rare cases to describe vascular calcinosis not related to CKD. This clarification is critical to advance the field in terms of pathologic diagnosis and treatment of CKD bone and mineral disorder. Factors that seem to promote the osteoblastic transformation of vascular smooth muscle cells and enhance deposition of calcium hydroxyapatite crystals include phosphorus activation of the Pit-1 receptor, bone morphogenic proteins 2 and 4, leptin, endogenous 1,25 dihydroxyvitamin D, vascular calcification activating factor, and measures of oxidative stress. These entities work to accelerate the atherosclerotic process in patients with CKD and may be future targets for diagnosis and treatment because randomized trials with hydroxymethylglutaryl-CoA reductase inhibitors have failed to attenuate the rate of progressive vascular calcification.

[Variance of expressions of extracellular matrix components and effects of anti-osteoporotic drugs on Mönckeberg's arteriosclerosis].

Mönckeberg-type arteriosclerosis occurs as a complication in diabetic, uremic patients and in postmenoposal women. It has been shown that arterial calcification generates loss of elasticity in tunica media. We have already reported that the expression of tropoelastin (TE), the precursor protein of elastin, is suppressed by arterial calcification, although no changes of mRNA expression of the other elastic fiber components, such as fibrillins, was observed. We examined the effects of bisphosphonates, known as anti-osteoporotic drugs, in inorganic phosphate (Pi)-induced calcified bovine aortic smooth muscle cells (BASMCs) (in vitro arterial calcification model). Treatment with the bisphosphonate risedronate, significantly inhibited calcium deposition in the arterial calcification model. Risedronate also inhibited suppression of TE mRNA expression and the progression of osteopontin (OPN) and core binding factor-alpha1 (Cbfa1), an osteogenic transcription factor, by BASMCs calcification. Basically, bisphosphonates could inhibit phenotypic transition such as SMC to osteoblast-like cell. Inhibitory effects of bisphosphonates were also shown in female Sprague-Dawley rats with calcinosis induced by administration of an over-dose of vitamin D2 (in vivo arterial calcification model). It is known that arterial calcification is accelerated by oxidative low-density lipoprotein (oxLDL). Therefore we examined the effects of 7-ketocholesterol (7kc), a component of oxLDL, on in vitro arterial calcification. Thereupon, it was revealed that 7kc drastically accelerated Pi-induced calcification, and risedronate completely restored the calcification and mRNA expression accelerated by 7kc.

Mönckeberg sclerosis revisited: a clarification of the histologic definition of Mönckeberg sclerosis.

CONTEXT: Medial calcification of muscular arteries is known as Mönckeberg sclerosis (MS). Although this was first described in 1903, disagreement persists over its precise histologic appearance. Some, including Mönckeberg, have written that the media alone are calcified, whereas others maintain that both the media and internal elastic lamina (IEL) are involved. Since vascular calcification is of great interest to investigators and clinicians, defined criteria for classifying calcified arterial lesions are important. OBJECTIVE: To clarify the histologic definition of MS with regard to calcification of the IEL. DESIGN: We reviewed slides from 14 incisional and excisional surgical biopsies and autopsy specimens containing arteries with previously diagnosed MS. We looked specifically for medial and IEL calcification and used von Kossa, alizarin red, and trichrome/elastic stains to confirm our findings. We also performed a literature search on the histologic appearance of MS. RESULTS: Both medial and IEL calcification were present in all specimens. Medial calcification extended alongside calcified IEL. In focal regions, calcification appeared limited to the IEL, with minimal medial calcification. Occasionally, calcified nodules in the media appeared separated from the IEL yet were connected to it in other planes of section. Despite these variations in appearance, IEL involvement was universal. Of 25 journal articles and texts, 10 state that MS involves the IEL with calcification, whereas 15 state or suggest that it does not. CONCLUSIONS: Our findings indicate MS involves both the IEL and media with calcification in spite of inconsistencies on this point in the medical literature.

Novel conformation-specific antibodies against matrix gamma-carboxyglutamic acid (Gla) protein: undercarboxylated matrix Gla protein as marker for vascular calcification.

OBJECTIVE: Matrix gamma-carboxyglutamic acid (Gla) protein (MGP), a vitamin K-dependent protein, is a potent in vivo inhibitor of arterial calcification. We hypothesized that low endogenous production of MGP and impaired carboxylation of MGP may contribute to the development or the progression of vascular disease. METHODS AND RESULTS: Novel conformation-specific antibodies against MGP were used for immunohistochemistry of healthy and sclerotic arteries. In healthy arteries, MGP was mainly displayed around the elastin fibers in the tunica media. The staining colocalized with that for carboxylated MGP, whereas undercarboxylated MGP (ucMGP) was not detected. In atherosclerotic arteries, ucMGP was found in the intima, where it was associated with vesicular structures. In Mönckeberg's sclerosis of the media, ucMGP was localized around all areas of calcification. The results indicate that ucMGP is strongly associated with vascular calcification of different etiologies. In a separate study, serum MGP concentrations in a cohort of 172 subjects who had undergone percutaneous coronary intervention were significantly reduced compared with an apparently healthy population. CONCLUSIONS: These data show that impaired carboxylation of MGP is associated with intimal and medial vascular calcification and suggest the essentiality of the vitamin K modification to the function of MGP as an inhibitor of ectopic calcification.