Medial Arterial Calcification Research Articles

Epigallocatechin-3-gallate inhibits osteogenic differentiation of vascular smooth muscle cells through the transcription factor JunB.

Medial arterial calcification (MAC) accompanying chronic kidney disease (CKD) leads to increased vessel wall stiffness, myocardial ischemia, heart failure, and increased cardiovascular morbidity and mortality. Unfortunately, there are currently no drugs available to treat MAC. The natural polyphenol epigallocatechin-3-gallate (EGCG) has been demonstrated to protect against cardiovascular disease; however, whether EGCG supplementation inhibits MAC in CKD remains unclear. In this study, we utilize a CKD-associated MAC model to investigate the effects of EGCG on vascular calcification and elucidate the underlying mechanisms involved. Our findings demonstrate that EGCG treatment significantly reduces calcium phosphate deposition and osteogenic differentiation of VSMCs in vivo and in vitro in a dose-dependent manner. In addition, through RNA sequencing (RNA-seq) analysis, we show a significant activation of the transcription factor JunB both in CKD mouse arteries and in osteoblast-like VSMCs. Notably, EGCG effectively suppresses CKD-associated MAC by inhibiting the activity of JunB. In addition, overexpression of JunB can abolish while knockdown of JunB can enhance the inhibitory effect of EGCG on the osteogenic differentiation of VSMCs. Furthermore, EGCG supplementation inhibits MAC in CKD via modulation of the JunB-dependent Ras/Raf/MEK/ERK signaling pathway. In conclusion, our study highlights the potential therapeutic value of EGCG for managing CKD-associated MAC, as it mitigates this pathological process through targeted inactivation of JunB.

A prospective observational study to evaluate a possible relationship between vitamin K antagonist therapy and risk of peripheral arterial disease in patients with type 2 diabetes.

The use of vitamin K antagonists (VKAs) may increase the risk of peripheral arterial disease (PAD) because vitamin K is a strong inhibitor of medial arterial calcification. Type 2 diabetes (T2D) exposes patients to an increased risk of PAD. We examined how the use of VKAs modulates the risk of incident PAD in people with T2D. SURDIAGENE is a French cohort including 1468 patients with T2D with a prospective follow-up from 2002 to 2015. The primary outcome of the current analysis was the first occurrence of PAD, a composite of lower-limb amputation (LLA) or lower-limb revascularization. LLA and lower-limb revascularization were considered individually as secondary outcomes. During a 7-year median follow-up, PAD occurred in 147 (10%) of the 1468 participants. The use of VKAs was not significantly associated with the risk of PAD [multivariable adjusted hazard ratio (HR) 1.42, 95% confidence interval (CI), 0.88-2.31]. During the study period, LLA and lower-limb revascularization occurred in 82 (6%) and 105 (7%) participants, respectively. The use of VKAs was significantly associated with increased risk of LLA [multivariable adjusted HR 1.90 (95% CI, 1.04-3.47)], but not lower-limb revascularization [multivariable adjusted HR 1.08 (95% CI, 0.59-1.97)]. In this prospective study, we did not observe any excess risk of PAD requiring lower-limb revascularization in people with type 2 diabetes using VKAs. However, our data suggest a high risk of LLA in VKA users. Further studies are required to confirm this observation.

FHL2 in arterial medial calcification in chronic kidney disease.

Arterial medial calcification (AMC) is a common complication in individuals with chronic kidney disease (CKD), which can lead to cardiovascular morbidity and mortality. The progression of AMC is controlled by a key transcription factor called runt-related transcription factor 2 (RUNX2), which induces vascular smooth muscle cells (VSMCs) transdifferentiation into a osteogenic phenotype. However, RUNX2 has not been targeted for therapy due to its essential role in bone development. The objective of our study was to discover a RUNX2 coactivator that is highly expressed in arterial VSMCs as a potential therapy for AMC. We employed transcriptomic analysis of human data and an animal reporter system to pinpoint FHL2 as a potential target. Subsequently, we investigated the mRNA and protein expression patterns of FHL2 in the aortas of both human and animal subjects with CKD. To examine the role of FHL2 in the RUNX2 transcription machinery, we conducted coimmunoprecipitation (Co-IP) and chromatin immunoprecipitation (ChIP) experiments. Next, we manipulated FHL2 expression in cultured VSMCs to examine its impact on high phosphate-induced transdifferentiation. Finally, we employed FHL2 null mice to confirm the role of FHL2 in the development of AMC in vivo. Among all the potential RUNX2 cofactor, FHL2 displays selective expression within the cardiovascular system. In the context of CKD subjects, FHL2 undergoes upregulation and translocation from the cytosol to the nucleus of arterial VSMCs. Once in the nucleus, FHL2 interacts structurally and functionally with RUNX2, acting as a coactivator of RUNX2. Notably, the inhibition of FHL2 expression averts transdifferentiation of VSMCs into an osteogenic phenotype and mitigates aortic calcification in uremic animals, without causing any detrimental effects on the skeletal system. These observations provide evidence that FHL2 is a promising target for treating arterial calcification in patients with CKD.

Mir-195-5p targets Smad7 regulation of the Wnt/β-catenin pathway to promote osteogenic differentiation of vascular smooth muscle cells

In this study, we sought to investigate the mechanisms of action of miR-195-5p in the osteogenic differentiation of vascular smooth muscle cells (VSMCs), and thereby provide novel insights and a reference for the targeted therapy of arterial media calcification. VSMC differentiation was induced using sodium β-glycerophosphate, and we investigated the effects of transfecting cells with miR-195-5p mimics, vectors overexpressing Smad7, and the Wnt/β-catenin pathway inhibitor (KYA1797K) on VSMC differentiation by determining cell viability and apoptosis, and the mRNA and protein expression of factors associated with osteogenic differentiation and the Wnt/β-catenin pathway. The results revealed that miR-195-5p mimics enhanced the osteogenic differentiation of VSMCs induced by β-glycerophosphate, whereas the overexpression of Smad7 reversed this phenomenon. In addition, KYA1797K was found to promote the effects of Smad7 overexpression. In conclusion, by targeting, Smad7, miR-195-5p promotes the Wnt/β-catenin pathway. and thus the osteogenic differentiation of VSMCs. These findings will provide a reference for elucidating the mechanisms whereby miR-195-5p regulates osteogenic differentiation.

β,γ-Methylene-ATP and its metabolite medronic acid affect both arterial media calcification and bone mineralization in non-CKD and CKD rats.

Arterial media calcification or pathological deposition of calcium-phosphate crystals in the vessel wall contributes significantly to the high mortality rate observed in patients with CKD. Extracellular nucleotides (ie, ATP or UTP) regulate the arterial calcification process by interacting with (1) purinergic receptors and (2) breakdown via ecto-nucleotidases, such as ectonucleotide pyrophosphatase/phosphodiesterase NPP1 or NPP3, affecting the local levels of calcification inhibitor, pyrophosphate, and stimulator inorganic phosphate (PPi/Pi ratio). Also, it has been shown that ATP analogs (ie, β,γ-methylene-ATP [β,γ-meATP]) inhibit vascular smooth muscle cell calcification in vitro. In the first experiment, daily dosing of β,γ-meATP (2mg/kg) was investigated in rats fed a warfarin diet to trigger the development of non-CKD-related arterial medial calcifications. This study showed that β,γ-meATP significantly lowered the calcium scores in the aorta and peripheral vessels in warfarin-exposed rats. In a second experiment, daily dosing of 4mg/kg β,γ-meATP and its metabolite medronic acid (MDP) was analyzed in rats fed an adenine diet to promote the development of CKD-related arterial medial calcification. Administration of β,γ-meATP and MDP did not significantly decrease aortic calcification scores in this model. Moreover, both compounds induced deleterious effects on physiological bone mineralization, causing an imminent risk for worsening the already compromised bone status in CKD. Due to this, it was not possible to raise the dosage of both compounds to tackle CKD-related arterial calcification. Again, this points out the difficult task of targeting solely ectopic calcifications without negatively affecting physiological bone mineralization. On the other hand, aortic mRNA expression of Enpp1 and Enpp3 was significantly and positively associated with aortic calcification scores, suggesting that normalizing the aortic NPP1/3 activity to control values might be a possible target to treat (CKD-induced) arterial media calcifications.

Orphan Nuclear Receptor NR4A3 Promotes Vascular Calcification via Histone Lactylation.

Medial arterial calcification is a chronic systemic vascular disorder distinct from atherosclerosis and is commonly observed in patients with chronic kidney disease, diabetes, and aging individuals. We previously showed that NR4A3 (nuclear receptor subfamily 4 group A member 3), an orphan nuclear receptor, is a key regulator in apo (apolipoprotein) A-IV-induced atherosclerosis progression; however, its role in vascular calcification is poorly understood. We generated NR4A3-/- mice and 2 different types of medial arterial calcification models to investigate the biological roles of NR4A3 in vascular calcification. RNA-seq was performed to determine the transcriptional profile of NR4A3-/- vascular smooth muscle cells under β-glycerophosphate treatment. We integrated Cleavage Under Targets and Tagmentation analysis and RNA-seq data to further investigate the gene regulatory mechanisms of NR4A3 in arterial calcification and target genes regulated by histone lactylation. NR4A3 expression was upregulated in calcified aortic tissues from chronic kidney disease mice, 1,25(OH)2VitD3 overload-induced mice, and human calcified aorta. NR4A3 deficiency preserved the vascular smooth muscle cell contractile phenotype, inhibited osteoblast differentiation-related gene expression, and reduced calcium deposition in the vasculature. Further, NR4A3 deficiency lowered the glycolytic rate and lactate production during the calcification process and decreased histone lactylation. Mechanistic studies further showed that NR4A3 enhanced glycolysis activity by directly binding to the promoter regions of the 2 glycolysis genes ALDOA and PFKL and driving their transcriptional initiation. Furthermore, histone lactylation promoted medial calcification both in vivo and in vitro. NR4A3 deficiency inhibited the transcription activation and expression of Phospho1 (phosphatase orphan 1). Consistently, pharmacological inhibition of Phospho1 attenuated calcium deposition in NR4A3-overexpressed vascular smooth muscle cells, whereas overexpression of Phospho1 reversed the anticalcific effect of NR4A3 deficiency in vascular smooth muscle cells. Taken together, our findings reveal that NR4A3-mediated histone lactylation is a novel metabolome-epigenome signaling cascade mechanism that participates in the pathogenesis of medial arterial calcification.

Pedal medial arterial calcification influences the outcomes of isolated infra-malleolar interventions for chronic limb-threatening ischemia

ObjectiveInframalleolar disease is present in most diabetic patients presenting with tissue loss. Inframalleolar (pedal) artery disease and pedal medial arterial calcification (pMAC) are associated with major amputation in patients with chronic limb-threatening ischemia (CLTI). This study aimed to examine the impact of pMAC on the outcomes after isolated inframalleolar (pedal artery) interventions. MethodsA database of lower extremity endovascular intervention for patients with tissue loss between 2007 and 2022 was retrospectively queried. Patients with CLTI were selected, and those undergoing isolated inframalleolar intervention on the dorsalis pedis and medial and lateral tarsal arteries and who had foot x-rays were identified. X-rays were assessed blindly for pMAC and scored on a scale of 0 to 5. Patients with concomitant superficial femoral artery and tibial interventions were excluded. Intention to treat analysis by the patient was performed. Amputation-free survival (survival without major amputation) was evaluated. ResultsA total of 223 patients (51% female; 87% Hispanic; average age, 66 years; 323 vessels) underwent isolated infra-malleolar intervention for tissue loss. All patients had diabetes, 96% had hypertension, 79% had hyperlipidemia, and 63% had chronic renal insufficiency (55% of these were on hemodialysis). Most of the patients had Wound, Ischemia, and foot Infection (WIfI) stage 3 disease and had various stages of pMAC: severe (score = 5) in 48%, moderate (score = 2-4) in 31%, and mild (score = 0-1) in 21% of the patients. Technical success was 94%, with a median of one vessel treated per patient. All failures were in severe pMAC. Overall, major adverse cardiovascular events was 0.9% at 90 days after the procedure. Following the intervention, most patients underwent a planned forefoot amputation (single digit, multiple digits, ray amputation, or trans-metatarsal amputation). WIfI ischemic grade was improved by 51%. Wound healing at 3 months was 69%. Those not healing underwent below-knee amputations. The overall 5-year amputation-free survival rate was 35% ± 9%. The severity of pMAC was associated with decreased AFS. ConclusionsIncreasing severity of pMAC influences the technical and long-term outcomes of infra-malleolar intervention in diabetes. Severe pMAC is associated with amputation and should be considered as a variable in the shared decision-making of diabetic patients with CLTI.

Smooth muscle cell-specific matrix metalloproteinase 3 deletion reduces osteogenic transformation and medial artery calcification.

Vascular calcification is highly prevalent in atherosclerosis, diabetes, and chronic kidney disease. It is associated with increased morbidity and mortality in patients with cardiovascular disease. Matrix metalloproteinase 3 (MMP-3), also known as stromelysin-1, is part of the large matrix metalloproteinase family. It can degrade extracellular matrix components of the arterial wall including elastin, which plays a central role in medial calcification. In this study, we sought to determine the role of MMP-3 in medial calcification. We found that MMP-3 was increased in rodent models of medial calcification as well as in vascular smooth muscle cells (SMCs) cultured in a phosphate calcification medium. It was also highly expressed in calcified tibial arteries in patients with peripheral arterial disease (PAD). Knockdown and inhibition of MMP-3 suppressed phosphate-induced SMC osteogenic transformation and calcification, whereas the addition of a recombinant MMP-3 protein facilitated SMC calcification. In an ex vivo organ culture model and a rodent model of medial calcification induced by vitamin D3, we found that MMP-3 deficiency significantly suppressed medial calcification in the aorta. We further found that medial calcification and osteogenic transformation were significantly reduced in SMC-specific MMP-3-deficient mice, suggesting that MMP-3 in SMCs is an important factor in this process. These findings suggest that MMP-3 expression in vascular SMCs is an important regulator of medial calcification and that targeting MMP-3 could provide a therapeutic strategy to reduce it and address its consequences in patients with PAD.

Risk Factors for Medial and Intimal Intracranial Internal Carotid Artery Calcification in Men and Women with Cardiovascular Disease: The UCC-SMART Study

Introduction: Calcifications of the intracranial internal carotid artery (iICA) can lead to an increased risk for stroke. Two types of iICA calcification are known: those affecting the tunica intima or the tunica media. In extracranial arteries, risk factors and calcification patterns are different in women and men, but little is known regarding the iICA. In this study, we aimed to identify sex-specific risk profiles and medications associated to intimal and medial iICA calcification in patients with cardiovascular disease (CVD). Methods: Participants of the UCC-SMART cohort undergoing a non-contrast head CT within 6 months from the study inclusion were considered (n = 475). Intimal or medial iICA calcification pattern was assessed using a previously histology-validated method. Sex-stratified associations between calcification pattern and cardiovascular risk factors, laboratory parameters, and medication use were calculated using Poisson regression analysis with robust standard errors. Results: Two hundred and four women and 271 men (age range 24–79 years) were included. 45.4% of men and 34.8% of women showed intimal iICA calcification, while 28.4% of men and 24.0% of women showed medial iICA calcification. Minimal or no iICA calcification was observed in 26.2% of men and in 41.2% of women (reference group). Older age was associated with both calcification patterns in women and men. In women, use of vitamin K antagonists and lipid-lowering drugs was associated to medial calcification, while systolic blood pressure and glucose levels were associated to intimal calcification. In men, current smoking was associated to intimal calcification. Conclusions: Women and men with CVD show differences in risk profiles and medication use associated to intimal and medial iCA calcification.

Early hemodynamics after tibial transverse transport in patients with nonarterial stenosis and arterial stenosis diabetic foot.

The diagnosis of peripheral arteriopathy in the diabetic foot is complicated by diabetes and its advanced complications. It has been found that diabetic foot can be categorized into arterial stenosis and non-arterial stenosis, both of which have significant differences in hemodynamic characteristics. To evaluate the early hemodynamic changes in diabetic foot patients with nonarterial stenosis and arterial stenosis treated by tibial transverse transport (TTT) using high-frequency color Doppler ultrasonography (HFCDU) and a laser Doppler flowmeter. Twenty-five patients with Wagner grades 3-5 diabetic foot ulcers were treated with TTT, and the wound healing time and rate were recorded. Patients were grouped according to the results of preoperative lower-extremity ultrasonography. Cases with ≥ 50% stenosis in any of the femoral, popliteal, posterior tibial, anterior tibial, and peroneal arteries of the affected limb were classified as the arterial stenosis group (n = 16); otherwise, they were classified as the nonarterial stenosis group (n = 9). Before and one month after surgery, HFCDU was used to evaluate the degree of lower limb artery lesions and hemodynamic changes in patients. The degree of femoral-popliteal atherosclerotic stenosis, the degree of vascular stenosis and occlusion of the lower-knee outflow tract, and the degree of medial arterial calcification were scored; the three scores were added together to obtain the total score of lower extremity arteriopathy. PeriScanPIM3, a laser Doppler flowmeter system, was used to detect alterations in plantar microcirculation before and 1 mo after surgery. Wound healing and hemodynamic indices were compared between the two groups. The wound healing time of the diabetic foot was significantly shorter in the nonarterial stenosis group than in the arterial stenosis group (47.8 ± 13 vs 85.8 ± 26, P < 0.05), and the wound healing rate of both groups was 100%. The preoperative total lower extremity arteriopathy scores were lower in the nonarterial stenosis group than those in the arterial stenosis group (18.89 ± 8.87 vs 24.63 ± 3.52, P < 0.05). The nonarterial stenosis group showed higher preoperative popliteal artery (POA) blood flow than the arterial stenosis group (204.89 ± 80.76 cc/min vs 76.75 ± 48.49 cc/min, P < 0.05). Compared with the baseline (before surgery), the postoperative POA blood flow of the affected limb in the nonarterial stenosis group decreased one month after surgery (134.11 ± 47.84 cc/min vs 204.89 ± 80.76 cc/min, P < 0.05), while that in the arterial stenosis group increased (98.44 ± 30.73 cc/min vs 61.69 ± 21.70 cc/min, P < 0.05). Although the POA blood flow in the arterial stenosis group was obviously improved one month after surgery, it was still lower than that in the nonarterial stenosis group (98.44 ± 30.73 cc/min vs 134.11 ± 47.84 cc/min, P < 0.05). The nonarterial stenosis group had higher preoperative plantar microcirculation than the arterial stenosis group (56.1 ± 9.2 vs 33.2 ± 7.5, P < 0.05); compared with the baseline, the plantar microcirculation in the arterial stenosis group was significantly improved one month after surgery (51.9 ± 7.2, P < 0.05), while that in the nonarterial stenosis group was reduced (35.9 ± 7.2, P < 0.05). Based on preoperative HFCDU findings, diabetic foot patients can be divided into two categories: Those with nonarterial stenosis and those with arterial stenosis, with obvious differences in hemodynamic changes in the early postoperative period between them. In the early stage after TTT, the blood flow volume and velocity and the plantar microcirculation perfusion of the affected limb of the diabetic foot with nonarterial stenosis decreased compared with the baseline, while those of the diabetic foot with arterial stenosis improved significantly compared with the baseline, although both had smoothly healed diabetic foot ulcers.

Better Detection of Peripheral Arterial Disease with Toe-Brachial Index Compared to Ankle-Brachial Index among Taiwanese Patients with Diabetic Kidney Disease

Background: Ankle-brachial index (ABI) is a simple method for diagnosing peripheral artery disease (PAD) but has limited reliability in patients with diabetes mellitus (DM) because of medial artery calcification. Our study aims to investigate whether the toe brachial index (TBI) or the cardio-ankle vascular index (CAVI) has a better detection over the ABI for diagnosing PAD in diabetic kidney disease (DKD). Materials and Methods: A cohort of 368 patients (mean age 68.59 ± 13.14 years, 190 males and 178 females) with type 2 DM underwent ABI, TBI, and CAVI measurements at our outpatient clinic. Results: Of all enrolled patients, the TBI is significant in evaluating PAD, especially in patients whose chronic kidney disease (CKD) stage 3a with adjusted odds ratio (AOR) = 6.50, 95% confidence interval (CI) 1.63–25.97, p = 0.0080, stage 3b AOR = 7.47, 95% CI 1.52–36.81, p = 0.0135, and stage 4–5 AOR = 20.13, 95% CI 1.34–94.24, p = 0.0116. CAVI is also significant in CKD stage 1 with AOR = 0.16, 95% CI 0.03–0.77, p = 0.0223, stage 2 with AOR = 0.18, 95% CI 0.04–0.74, p = 0.0180, and stage 3a AOR = 0.31, 95% CI 0.10–0.93, p = 0.0375. Conclusion: TBI has a better yield of detection of PAD compared to ABI among Taiwanese patients with DKD. CAVI may play a role in the early stage of DKD.

Diagnostic accuracy of the maximal systolic acceleration to detect peripheral arterial disease

BackgroundDiagnosing peripheral arterial disease (PAD) can be challenging owing to medial arterial calcification (MAC) in patients with diabetes mellitus (DM) and chronic kidney disease (CKD). Current bedside tests, such as the ankle-brachial index and toe-brachial index, are often insufficient. The maximal systolic acceleration (ACCmax) is a velocimetric Doppler-derived parameter and could be a new promising test in the diagnostic workup of these patients. The primary aim of this study was to evaluate the diagnostic performance of the ACCmax to detect PAD. MethodsA retrospective cohort study was performed in a tertiary referral hospital. Patients ≥18 years old with suspected PAD who underwent ACCmax measurement(s) along with computed tomography angiography of the abdominal aorta and lower extremities (reference test) were eligible for inclusion. ACCmax measurements of the posterior tibial artery, anterior tibial artery and peroneal artery were collected. Diagnostic performance was assessed by using sensitivity, specificity, positive likelihood ratio, negative likelihood ratio, and area under the curve (AUC). ResultsIn total, 340 patients (618 limbs) were included. Approximately 40% suffered from DM and 30% had CKD. Diagnostic performance of the ACCmax to detect PAD for the posterior tibial artery showed a sensitivity of 90%, specificity of 93%, positive likelihood ratio of 12.83, and negative likelihood ratio of 0.11 (AUC, 0.953). For the anterior tibial artery, these results were 94%, 97%, 32.06, and 0.06 (same sequence as presented before) with an AUC of 0.984. The peroneal artery had a performance of 86%, 89%, 7.51, and 0.16, respectively (AUC, 0.893). Diagnostic accuracy of the ACCmax did not diminish in subgroup analysis for patients with DM or CKD. ConclusionsThe ACCmax showed excellent diagnostic performance to detect PAD, independent of patients prone to medial arterial calcification.

Screening for Peripheral Artery Disease Using an Automated Four-Limb Blood Pressure Monitor Equipped with Toe-Brachial Index Measurement.

Toe-brachial index (TBI) measurement helps to detect peripheral artery disease (PAD) in patients with incompressible ankle arteries due to medial arterial calcification, which is most frequently associated with diabetes. We aimed to evaluate how an automated four-limb blood pressure monitor equipped with TBI measurement could contribute to PAD screening. In 117 patients (mean age 63.2 ± 12.8 years), ankle-brachial index (ABI) measurement was performed using the Doppler-method and the MESI mTablet. TBI was obtained via photoplethysmography (MESI mTablet, SysToe) and a laser Doppler fluxmeter (PeriFlux 5000). Lower limb PAD lesions were evaluated based on vascular imaging. A significant correlation was found between Doppler and MESI ankle-brachial index values (r = 0.672), which was stronger in non-diabetic (r = 0.744) than in diabetic (r = 0.562) patients. At an ABI cut-off of 0.9, Doppler (AUC = 0.888) showed a sensitivity/specificity of 67.1%/97.4%, MESI (AUC 0.891) exhibited a sensitivity/specificity of 57.0%/100%; at a cut-off of 1.0, MESI demonstrated a sensitivity/specificity of 74.7%/94.8%. The TBI values measured using the three devices did not differ significantly (p = 0.33). At a TBI cut-off of 0.7, MESI (AUC = 0.909) revealed a sensitivity/specificity of 92.1%/67.5%. Combining MESI ABI and TBI measurements recognised 92.4% of PAD limbs. Using an ABI cut-off level of 1.0 and sequential TBI measurement increases the sensitivity of the device in detecting PAD. The precise interpretation of the obtained results requires some expertise.

Antihypertensive treatment with hydrochlorothiazide-hydralazine combination aggravates medial vascular calcification in CKD rats with mineral bone disorder.

Arterial stiffness and medial vascular calcification, leading to isolated systolic blood pressure (BP), are major cardiovascular risk factors in patients with chronic kidney disease (CKD) and mineral bone disorders (MBD). The impact of BP on MBD-induced medial vascular calcification in CKD remains uncertain. We investigated whether BP reduction improves arterial stiffness and medial vascular calcification in a rat model of CKD-MBD. CKD was induced in Wistar rats by subtotal nephrectomy. Then, MBD was generated by a Ca/P-rich diet with calcitriol supplementation to induce medial vascular calcification. Two antihypertensive treatments were evaluated: (1) the angiotensin AT1 receptor antagonist losartan, and (2) the combination of the thiazide diuretic hydrochlorothiazide and the direct vasodilator hydralazine (HCTZ/HY). After 5 weeks, mean BP (MBP), pulse pressure (PP), and pulse wave velocity (PWV) were determined. Vascular calcification was assessed in the thoracic aorta. While MBP was similar in CKD-MBD and control CKD rats, PP and PWV were increased in CKD-MBD rats. The heightened arterial stiffness in CKD-MBD rats was associated with diffused medial calcification along the thoracic aorta. Although both losartan and HCTZ/HY reduced MBP in CKD-MBD rats, losartan did not affect PP and PWV nor medial vascular calcification, whereas HCTZ/HY, unexpectedly, further increased arterial stiffness and medial vascular calcification. In the rat model of CKD-MBD, antihypertensive treatment with losartan did not affect arterial stiffness or medial vascular calcification. However, HCTZ/HY treatment aggravated arterial stiffness and vascular calcification despite a similar reduction of MBP, suggesting a blood pressure-independent mechanism for vascular calcification.

Pedal Medial Arterial Calcification Influences Outcomes of Isolated Inframalleolar Interventions

Pedal Medial Arterial Calcification Influences Outcomes of Isolated Inframalleolar Interventions

Ultrasound-detected tibial artery calcification as a marker of cardiovascular and lower-limb risk in asymptomatic patients with type-2 diabetes.

Medial arterial calcification (MAC) is a vascular disease distinct from atherosclerosis. Recently, several studies have demonstrated that MAC is an important marker of cardiovascular events. We aim to assess the presence of MAC during ultrasound screening of lower-limb vasculature and its association with both cardiovascular (CV) and lower-limb events in patients with type-2 diabetes. A retrospective cohort study was conducted on 1119 patients with type-2 diabetes free from CV disease. A CV work-up, including vascular ultrasound, was performed for each patient. The presence of MAC was assessed on posterior tibial arteries and ankle-brachial index (ABI) was measured. Major acute CV events (MACEs) and lower-limb events (MALEs) were recorded as a composite endpoint for a 5-year period. We identified MAC among 212 (18.9%) patients. The independent determinants of MAC were age and diabetic retinopathy. Over a period of 5 years, 125 MACEs and 22 MALEs occurred. MAC was significantly associated with the composite outcome MACE + MALE (HR = 1.94; 95% CI: 1.23, 3.08, p = 0.005) or with MACE (HR = 1.85; 95% CI: 1.16, 2.95, p = 0.010). Adjusted for ABI and diabetic foot wound, MAC remained a determinant of MALE (HR = 5.49; 95% CI: 2.19, 13.76, p < 0.001). Considering each ABI group, MAC was associated with both MACE and MALE in the normal ABI group. Ultrasound-detected MAC on tibial arteries seems to be a determinant of both CV and lower-limb events, independent from ABI. MAC helps to refine the CV risk in patients with normal ABI.

Medial arterial calcification score is associated with increased risk of major limb amputation

ObjectiveThe pedal medial arterial calcification (MAC) score has been associated with risk of major limb amputation in patients with chronic limb-threatening ischemia. This study aimed to validate the pedal MAC scoring system in a multi-institutional analysis to validate its usefulness in limb amputation risk prediction. MethodsA multi-institution, retrospective study of patients who underwent endovascular or open surgical infrainguinal revascularization for chronic limb-threatening ischemia was performed. MAC scores of 0 to 5 were assigned based on visible calcified arteries on foot X ray then trichotomized (0-1, 2-4, 5) for analysis. The primary outcome was major limb amputation at 6 months. Adjusted Kaplan-Meier models were used to analyze time-to-major amputation across groups. ResultsThere were 176 patients with 184 affected limbs (mean age, 66 years; 61% male; 60% White), of whom 97% presented with a wound. The MAC score was 0 in 41%, 1 in 9%, 2 in 13%, 3 in 11%, 4 in 13%, and 5 in 13% of the limbs. There were 26 major amputations (14%) and 16 deaths (8.7%) within 6 months. Patients with MAC 5 had a significantly higher risk of major limb amputation than both the 0 to 1 and 2 to 4 groups (P = .001 and P = .044, respectively), and lower overall amputation-free survival (log-rank P = .008). ConclusionsPedal MAC score is a reproducible and generalizable measure of inframalleolar arterial disease that can be used with Wound, Ischemia, and foot Infection staging to predict major limb amputation in patients with chronic limb-threatening ischemia.

Cold exposure protects against medial arterial calcification development via autophagy

Medial arterial calcification (MAC), a systemic vascular disease different from atherosclerosis, is associated with an increased incidence of cardiovascular events. Several studies have demonstrated that ambient temperature is one of the most important factors affecting cardiovascular events. However, there has been limited research on the effect of different ambient temperatures on MAC. In the present study, we showed that cold temperature exposure (CT) in mice slowed down the formation of vitamin D (VD)-induced vascular calcification compared with room temperature exposure (RT). To investigate the mechanism involved, we isolated plasma-derived exosomes from mice subjected to CT or RT for 30 days (CT-Exo or RT-Exo, respectively). Compared with RT-Exo, CT-Exo remarkably alleviated the calcification/senescence formation of vascular smooth muscle cells (VSMCs) and promoted autophagy by activating the phosphorylation of AMP-activated protein kinase (p-AMPK) and inhibiting phosphorylation of mammalian target of rapamycin (p-mTOR). At the same time, CT-Exo promoted autophagy in β-glycerophosphate (β-GP)-induced VSMCs. The number of autophagosomes and the expression of autophagy-related proteins ATG5 and LC3B increased, while the expression of p62 decreased. Based on a microRNA chip microarray assay and real-time polymerase chain reaction, miR-320a-3p was highly enriched in CT-Exo as well as thoracic aortic vessels in CT mice. miR-320a-3p downregulation in CT-Exo using AntagomiR-320a-3p inhibited autophagy and blunted its anti-calcification protective effect on VSMCs. Moreover, we identified that programmed cell death 4 (PDCD4) is a target of miR-320a-3p, and silencing PDCD4 increased autophagy and decreased calcification in VSMCs. Treatment with CT-Exo alleviated the formation of MAC in VD-treated mice, while these effects were partially reversed by GW4869. Furthermore, the anti-arterial calcification protective effects of CT-Exo were largely abolished by AntagomiR-320a-3p in VD-induced mice. In summary, we have highlighted that prolonged cold may be a good way to reduce the incidence of MAC. Specifically, miR-320a-3p from CT-Exo could protect against the initiation and progression of MAC via the AMPK/mTOR autophagy pathway.Graphic

Extracellular vesicles in atherosclerosis and vascular calcification: the versatile non-coding RNAs from endothelial cells and vascular smooth muscle cells.

Atherosclerosis (AS) is characterized by the accumulation of lipids, fibrous elements, and calcification in the innermost layers of arteries. Vascular calcification (VC), the deposition of calcium and phosphate within the arterial wall, is an important characteristic of AS natural history. However, medial arterial calcification (MAC) differs from intimal calcification and cannot simply be explained as the consequence of AS. Endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) are directly involved in AS and VC processes. Understanding the communication between ECs and VSMCs is critical in revealing mechanisms underlying AS and VC. Extracellular vesicles (EVs) are found as intercellular messengers in kinds of physiological processes and pathological progression. Non-coding RNAs (ncRNAs) encapsulated in EVs are involved in AS and VC, including microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs). The effects of ncRNAs have not been comprehensively understood, especially encapsulated in EVs. Some ncRNAs have demonstrated significant roles in AS and VC, but it remains unclear the functions of the majority ncRNAs detected in EVs. In this review, we summarize ncRNAs encapsulated in EC-EVs and VSMC-EVs, and the signaling pathways that are involved in AS and VC.

Treatment effect of medial arterial calcification in below-knee after Auryon laser atherectomy using micro-CT and histologic evaluation

PurposeTo determine the frequency of medial arterial calcification (MAC) fracture following Auryon laser atherectomy using micro-CT and histologic evaluation in an atherosclerotic human cadaveric limb model. MethodsTwo below-the-knee calcified arterial segments from human cadaver limbs underwent treatment with the Auryon laser system with or without plain old balloon angioplasty (POBA). Micro-CT and angiography were performed before and after treatment followed by histological evaluation of regions showing calcium disruption. ResultsAll treatment zones were successfully treated with the Auryon laser (n = 9). Six of 9 treatment zones showed calcium fracture on micro-CT. Each treatment zone was further subdivided using micro-CT analysis (36 evaluated sections) of which 18 sections revealed calcium fracture. Sections with calcium fracture had significantly more confluent and uninterrupted circumferential calcification than sections without calcium fracture (arc of calcification 360.0 [323.7–360.0] vs 312.8 [247.4–314.2] degree, p = 0.007), whereas there were no differences in size of calcium burden (3.4 [2.8–3.9] vs 2.8 [1.3–4.6] mm2, p = 0.46). No arterial dissection or rupture was seen. ConclusionsAuryon laser atherectomy produced fractures of medial arterial calcification in this cadaveric human atherosclerotic peripheral artery model. This effect was observed in arterial segments with a pattern of circumferential uninterrupted calcification (i.e. larger arc of calcification) regardless of calcium burden. Our pilot data suggests Auryon laser may be a promising therapy for calcified lesions.