Untitled Document
Vascular calcification (VC), which is categorized by intimal and medial calcification, depending on the site(s) involved within the vessel, is closely related to cardiovascular disease. Specifically, medial calcification is prevalent in certain medical situations, including chronic kidney disease and diabetes. The past few decades have seen extensive research into VC, revealing that the mechanism of VC is not merely a consequence of a high-phosphorous and -calcium milieu, but also occurs via delicate and well-organized biologic processes, including an imbalance between osteochondrogenic signaling and anticalcific events. In addition to traditionally established osteogenic signaling, dysfunctional calcium homeostasis is prerequisite in the development of VC. Moreover, loss of defensive mechanisms, by microorganelle dysfunction, including hyper-fragmented mitochondria, mitochondrial oxidative stress, defective autophagy or mitophagy, and endoplasmic reticulum (ER) stress, may all contribute to VC. To facilitate the understanding of vascular calcification, across any number of bioscientific disciplines, we provide this review of a detailed updated molecular mechanism of VC. This encompasses a vascular smooth muscle phenotypic of osteogenic differentiation, and multiple signaling pathways of VC induction, including the roles of inflammation and cellular microorganelle genesis.
1. Introduction
Vascular calcification (VC) is defined as mineral deposition, in the vasculature, in a form of calcium-phosphate complexes. Although VC is regarded as part of the normal aging process, certain pathological processes such as diabetes, hypertension, chronic kidney disease (CKD), and or rare hereditary disorders, may also precipitate the condition.
Traditionally, calcification is classified into two forms, depending on where the mineral is deposited. Intimal calcification is closely related to lipid deposits, and the clinically relevant infiltration of inflammatory cells, with obstructive arterial disease, whereas the latter is more pronounced by transformation into osteoblast-like cells from smooth muscle cells. Medial calcification is more prevalent in the aforementioned diseases, including CKD, diabetes, and arterial stiffness, rather than obstructions that are clinically significant in this pathology.
VC has an inseparable relationship with atherosclerotic vascular disease. In addition, it is known that arterial stiffness, which represents the functional disturbance of VC, is an independent predictor of cardiovascular mortality. Loss of elastin is coupled with medial calcification, and the degradation of elastin is believed to further contribute to the osteogenic process in aortic tissue. Elastin is the most abundant protein in the walls of the arteries, and thus its loss increases medial calcification, increased pulse pressure, left ventricular hypertrophy, and eventually, cardiovascular death. A recent report showed that monitoring of 10-year ambulatory blood pressure changes in older hypertensives revealed that 24-h pulse pressure better predicts mortality than 24-h systolic blood pressure. This finding suggests that in clinical perspective, reduction of arterial stiffness should be underscored as much as lowering systolic blood pressure.
In addition, despite continuous attempts, caclciphylaxis and ectopic calcification remains a clinical unmet need in certain clinical settings. As a result of recent advances in the field of VC pathogenesis, our current understanding of the mechanism of VC encompasses chronic inflammation, autophagy defects, endoplasmic reticulum (ER) stress, and mitochondrial dysfunction and its dynamics.
In this review, we summarize traditionally well-known mechanisms of VC, followed by recent updates regarding VC pathogenesis.
2. Classification of Vascular Calcification, Depending on Site and Etiology
VC is caused by the deposition of pathological mineral in the vascular system. Calcification has been classified, depending on where the mineral was deposited. VC of the vessel wall occurs in the intimal and medial layers. It can also be found in valvular calcification and calciphlaxis. Intimal calcification is associated with atherosclerotic plaques and is known as the result of lipid accumulation, macrophage invasion, proliferation of smooth muscle cells, and dysfunction of extracellular matrix proteins, in response to chronic arterial inflammation. These unstable and rupturable plaques form on the inner blood vessel wall, causing obstructive vascular disease.
In atherosclerotic neointima, calcification is initiated as microcalcifications, which, when present in the vascular wall, might play a pivotal role in overt atherosclerotic plaques. Microcalcification is defined as small calcium deposit (< 5 µm), initiated by necrotic or apoptotic cell death within lipid core. This type of calcification is observed in high-risk plaque. If the rupture of the plaque is perturbed due to the subsequent thrombotic occlusion, healing of the necrotic core and subsequent formation of macrocalcification (> 5 µm) starts, resulting in plaque stabilization.
This process is believed to be derived from apoptotic smooth muscle cells (SMCs), or matrix vesicles (“exosomes”) released by these cells near the internal elastic lamina. This occurs before changes in the intimal content of calcification-regulation proteins, such as osteocalcin (OC) and bone morphogenic protein (BMP) 2, but coincided with enhanced expression of uncarboxylated matrix gla protein (MGP). Increased nucleation sites may facilitate the precipitation of Ca2+ salts at the micrometer scale. Early-stage microcalcifications play a pathological role in the onset and progression of vascular disease.
Medial calcification is associated with aging, diabetes mellitus, hypertension, osteoporosis, and CKD. It can occur even without vascular stenosis, which mainly causes vascular stiffness, and increases the incidence of cardiovascular complications. The medial layer of the vessel wall is composed of smooth muscle cells and the elastin-rich extracellular matrix. In medial calcification, the process of differentiation of smooth muscle cells (SMCs) into osteoblast-like cells is akin to bone formation, and is related to genes such as BMP2, Msh Homeobox 2 (MSX2), and alkaline phosphatase (ALP).
It is also known that this differentiation begins with calcium deposition, through the production of matrix vesicles produced by SMCs. This phenomenon becomes possible by reducing calcification inhibitors, increasing oxidant or endoplasmic reticulum (ER) stress, during SMC signaling, apoptosis, and disorder of calcium-phosphate homeostasis associated with DNA damage, resulting from abnormal system hormonal regulation.
Arterial stiffness and medial calcification are independent predictors of cardiovascular mortality, and have been strongly and reciprocally correlated in many clinical studies. Each process intensifies each other, to create a vicious cycle. Here, hypertension plays a critical role in this vicious cycle. Hypertension promotes extracellular remodeling by accelerating type 1 collagen, fibronectin, and proteoglycans accumulation primarily in intima media. In addition, aberrant elastin production and deposition are accompanied. Newly synthesized fibers under pathological stimuli are less effective, therefore qualitative abnormalities of elastin occur. In addition, elastin-rich extracellular matrix (ECM) is degraded and causes accumulation of elastin-derived peptides. This cascade licenses a phenotypic switch of SMC. SMC endowed with proliferative and migratory activity plays a critical role of osteogenic differentiation of vascular cell. This pathological environment induces VC which further accelerates vascular stiffness and increase of pulse pressure. This phenomenon is observed in clinical situation. Compared with normotensive patient, patients with isolated systolic hypertension manifested with increased calcification of abdominal and descending thoracic aorta.
2.1. Valvular Calcification
Aortic valvular calcification, aortic sclerosis, and aortic stenosis, seen in different stages of calcific aortic valvular disease share a common pathophysiological process. The main cause of calcific aortic valvular disease is age-related degenerative process. In addition, genetic predisposition such as IL-10, Lp(a), or apolipoprotein B polymorphism or congenital valvular defects can cause valvular calcification.
Traditional risk factors of atherosclerosis as arterial hypertension, kidney failure, male sex, diabetes, and dyslipidemia are also regarded as a risk factor of early-onset valvular calcification. When severe valvular calcification results in aortic valve narrowing, it is called aortic stenosis. If the aortic valve becomes narrowed and severely dysfunctional, replacement surgery is required. In this case, aortic calcification can be seen as an early sign of heart disease, thus preemptive efforts to prevent the development of serious conditions by inhibition of calcification are required.
2.2. Calciphylaxsis
Calciphylaxis is a clinical resultant syndrome of arteriolar calcification, commonly investigated in end-stage renal disease patients on dialysis. It is induced by intense deposition of calcium accompanied by intimal proliferation, fibrosis, and thrombosis. These processes eventually lead to necrosis or ischemia in small blood vessels, skin, and other organs. Risk factors for calciphylaxis are high calcium-phosphate product, elevated level of parathyroid hormone, hypoalbuminemia, diabetes, female sex, obesity, and warfarin overdose. This disease is rare, but fatal and even if it is diagnosed at an early stage, the mortality rate is exceptionally high and the success rate of healing is low. The exact cause of calciphylaxis is still unknown, but its pathology includes tunica medial calcification, necrosis of tissue. It is common in ESRD patients, therefore it is likely that intimal hyperplasia and medial calcification is entangled with etiology.
3. Vascular Smooth Muscle Cell Phenotypic Differentiation in High-Phosphate Environments
Disruption of mineral homeostasis and high phosphate levels are considered to be the main determinants of VC in CKD. Hyperphosphatemia often occurs as a result of renal failure. However, the effect on calcification of the phosphate binder is not apparent. This is presumably due to the intracellular system that regulates phosphate, and the availability of bone to supply phosphate.
It is well accepted that phosphate complexes activate pro-calcific intracellular signaling pathways. Increased levels of calcium phosphate products associate with the development of vascular calcification in CKD. Even when renal function is normal, increased phosphate in serum associates with cardiovascular mortality and coronary artery calcification, suggesting that phosphate plays an important role in the pathophysiology of VC.
The mechanism of initiation and progression of VC is similar to the phenomenon of physiological bone formation. High phosphate upregulates Pit1 to raise intracellular levels of inorganic phosphate (Pi), inducing runt-related transcription factor 2 (RUNX2). This enhances the osteogenic transition of vascular SMC (VSMC). Downregulation of calcification inhibitors, release of extracellular vesicles, and remodeling of extracellular matrix, as well as osteo-/chondrogenic transdifferentiation and apoptosis of vascular cells contribute to this pathology. VSMCs play a key role in this phenomenon, and the processes are not mutually exclusive.
Calcium and phosphate concentrations exceed their solubility under pathological conditions, and endogenous calcification inhibitors are required to prevent ectopic precipitation of calcium and phosphate. High extracellular phosphate levels inhibit the production of calcification inhibitors, and promote the production of exosomal vesicles lacking such inhibitors.
Hyperphosphatemia also induces extracellular matrix remodeling. The production of matrix metalloproteinases and cysteine proteases results in the degradation of matrix proteins, generation of bioactive elastin peptides, and the synthesis of collagen, creating a collagen-enriched ECM. In addition, hyperphosphatemia induces the expression of enzymes that regulate collagen crosslinking, and supramolecular organization. These events combine to cause extracellular matrix remodeling, which is involved in vascular mineralization.
Under high-phosphate circumstances, apoptosis or necrosis of VSMCs is induced, possibly generating apoptotic bodies from VSMCs, which could serve as nidi for calcium phosphate precipitation. Phosphate-regulated intracellular signaling includes Wnt/β-catenin, protein kinase B (PKB or Akt), nuclear factor-kappa B (NF-κB), and serum- and glucocorticoid-inducible kinase 1 (SGK1). The role of the inflammasome is also important. In addition, oxidative stress, caused by an imbalance of antioxidants and reactive oxygen species (ROS), is related to osteoinductive signals and apoptosis, and may be regulated by the Gas6/Axl pathway, Akt, AMP-activated protein kinase (AMPK), etc., in which phosphate directly regulates apoptosis.
In addition, it is known that vascular calcification affects intracellular signaling, due to epigenetic phenomena, including altered microRNA levels, DNA methylation, and histone modifications. In addition, aging affects calcification through osteoinductive signaling, inflammatory cytokines, and oxidative stress. Moreover, cellular phenomena that affect osteoinductive intracellular signaling, via hyperphosphatemia, include autophagy, ER stress, and mitochondrial dysfunction. Eventually, various signaling pathways trigger phosphate-induced osteo-/chondrogenic transdifferentiation of VSMCs, as associated with VC. These complex and cross-talking mechanisms closely support the postulate that VC is affected by phosphate levels. Finding critical pathways among these will be an important approach in treating VC.
In addition to changing the properties of VSMCs, formation of substrates that can cause calcium deposition, and the alteration of defenses to remove them, are known to be important factors in initiating or worsening calcification. Representative causes of deposition include apoptosis, autophagy inhibition, matrix vesicle production, and increased bone mineralization, due to increased bone resorption.
4. Main Factors Causing Vascular Calcification
4.1. Extracellular Vesicles
Extracellular vesicles are secreted, membrane-enclosed particles, such as microvesicles, matrix vesicles, multivesicular bodies, ectosomes, exosomes, microparticles, and apoptotic bodies. The inner core of extracellular vesicles consists of soluble proteins, lipids, and noncoding RNAs. Based on their mechanism of biogenesis, extracellular vesicles are classified into three types: exosomes, microvesicles and apoptotic bodies. Extracellular vesicles can transfer functional transcripts and lipids to target cells, triggering changes in the target cell’s phenotype. EVs secreted into body fluids, and microenvironments from other cell types, act in adjacent regions or in distant cells.
Extracellular vesicles are found in calcified human aortic valves, aortic media, and atherosclerotic intimal plaque. Pathological extracellular vesicles are secreted by SMCs, stromal cells, and macrophages, all in vessel walls. In the normal state, contractile VSMCs release extracellular vesicles, especially matrix vesicles, to maintain homeostasis. MVs contain vitamin K-dependent matrix GLA protein (MGP) and circulating fetuin-A (Fet-A). However, under pathological states, VSMCs transform into a synthetic phenotype to promote the secretion of matrix vesicles, and transform target cells into a calcified state. Moreover, SMC-derived calcifying extracellular vesicles aggregate and form microcalcifications, themselves. Large calcifications are formed by the accumulation of microcalcifications, and maturation of minerals.
Collagen acts as a scaffold in controlling size and shape during this growth process. However, in pathological environments (e.g., CKD (hyperphosphatemia), inflammation-driven atherosclerosis), VSMCs release extracellular vesicles, which contain more calcification-associated markers, and less calcification inhibitors. In atherosclerotic plaques, collagen receptor-deficient VSMCs show increased release of calcified extracellular vesicles, and precipitation of collagen and minerals. In this situation, transported proteins, in calcified EVs, possess the ability to uptake Ca2+ and inhibit Fet-A activity. Calcified extracellular vesicles may then induce osteogenic genes (RUNX2, SMAD1, osterix (SP7), tissue non-specific alkaline phosphatase (TNAP), and proinflammatory genes.
It has been suggested that VC, in CKD, associates with increased deposition of VSMC-derived vesicles. In one study, using electron microscopy, matrix vesicles contained calcium phosphate crystals that were deposited in the ECM, in patients with predialysis or dialysis, whereas MVs were not observed in healthy individuals.
Calcium-carrying EVs are regarded as an adaptive reaction because they extrude calcium from the cell, to prevent excess accumulation of intracellular calcium. In response to calcium overload, or calcifying stimuli, EV release initially occurs until eventually, the deletion of the calcification inhibitor causes the EV to change into calcified extracellular vesicles. Moreover, inhibition of sphingomyelin phosphodiesterase 3 (SMPD3), an important player in extracellular vesicles production, prevents extracellular vesicles secretion and VSMC calcification, suggesting that extracellular vesicles could be a therapeutic target for VC.