Valve Calcification Research Articles

Elucidating common biomarkers and pathways of osteoporosis and aortic valve calcification: insights into new therapeutic targets.

Osteoporosis and aortic valve calcification, prevalent in the elderly, have unclear common mechanisms. This study aims to uncover them through bioinformatics analysis. Microarray data from GEO was analyzed for osteoporosis and aortic valve calcification. Differential expression analysis identified co-expressed genes. SVM-RFE and random forest selected key genes. GO and KEGG enrichment analyses were performed. Immunoinfiltration and GSEA analyses were subsequently performed. NetworkAnalyst analyzed microRNAs/TFs. HERB predicted drugs, and molecular docking assessed targeting potential. Thirteen genes linked to osteoporosis and aortic valve calcification were identified. TNFSF11, KYNU, and HLA-DMB emerged as key genes. miRNAs, TFs, and drug predictions offered therapeutic insights. Molecular docking suggested 17-beta-estradiol and vitamin D3 as potential treatments. The study clarifies shared mechanisms of osteoporosis and aortic valve calcification, identifies biomarkers, and highlights TNFSF11, KYNU, and HLA-DMB. It also suggests 17-beta-estradiol and vitamin D3 as potential effective treatments.

Abstract 4140121: Lipoprotein(a) Local Sybthesis And Plasma Uptake Via MDFS5 in Human Calcified Valve

Background: High circulating levels of lipoprotein(a) [Lp(a)] are associated with increased risk of calcific aortic stenosis (CAS). Targeting Lp(a) metabolism holds promise for therapeutic interventions. However, the precise mechanisms by which Lp(a) accumulates on aortic valves still not fully understood. Hypothesis: Lp(a) could be synthesized in situ in human aortic valves under specific stimuli, such as calcification, in addition to being taken up from plasma via membrane receptors, including the newly identified receptor Major Facilitator Superfamily Domain Containing 5 (MFSD5). Methods: Immunohistochemistry was used to detect the Lp(a) and its receptor MFSD5 in human aortic valves from CAS patients or controls, which were non-CAS patients. Plasma Lp(a) levels were measured in individuals undergoing aortic valve resection. Human valvular interstitial cells (VICs) were cultured in procalcifying medium, with or without Lp(a) (10uM) for 14 and 21 days. Von-kossa or Alizarin Red staining were used to assess valve calcification. Expression of Lp(a) and MFSD5 was analyzed with quantitative RT-PCR and western blot. Results: Lp(a) was nearly absent in control valve site, whereas it was abundantly present in valve site from CAS patients (p＜0.05). The higher the level of Lp(a) detected, the greater the extent of calcification noted in valve site. The mRNA and protein levels of Lp(a) extracted from calcified human valves were significantly higher compared to controls. However, the concentration of plasma Lp(a) did not affect the expression of Lp(a) in valve site (p＞0.05). In vitro studies showed that Lp(a) mRNA was minimally detectable in normal VICs, but was significantly upregulated following exposure to procalcifying medium (p < 0.05). Lp(a) incubation led to severer calcification than that induced by procalcifying medium alone (p < 0.05). As MFSD5 was a newly identified receptor of Lp(a), we also test its expression. MDFS5 was increased in human calcified valve (p＜0.05) and calcified VICs (p＜0.05). These results showed that the Lp(a) in valve could be increased by either uptake from serum or by synthesized locally by proper stimuli. Conclusion: Our study has shown that the degree of aortic valve calcification is correlated with the level of Lp(a) in the valve site rather than in serum. We also verified MFSD5 was the receptor of Lp(a) on human valve. Targeting Lp(a) accumulation at the valve site may offer a more effective strategy for the prevention or treatment of CAS.

Abstract 4134841: A Diagnostic Pitfall: Subclavian Stenosis Mimicking Severe Aortic Stenosis on Echocardiography"

Background: Aortic stenosis (AS) is a common and serious valve disease in the elderly, often diagnosed using echocardiography. However, certain conditions can mimic AS, leading to diagnostic challenges. This case highlights the importance of a comprehensive vascular assessment in patients with atypical symptoms and echocardiographic findings suggestive of severe AS. Case Presentation: An 80-year-old female presented with progressive dizziness and double vision over the past few months. She reported a long-standing history of lower blood pressure in her right arm compared to her left. Her medical history was significant for HER2-positive intraductal breast cancer treated with neoadjuvant chemotherapy. An echocardiogram performed two months prior to presentation, using a Pedoff probe, indicated severe AS with a peak systolic velocity of 5.1 m/sec, a mean systolic gradient of 50 mm Hg, and an aortic valve area of 0.6 cm. Investigations: Given the discrepancy in blood pressure between her arms and the wide open aortic valve on the echo which dosen't corrleate with the doppler numbers, CTA neck and chest was performed, revealing a dense calcific plaque causing severe stenosis at the origin of the left subclavian artery. An angiogram confirmed >75% stenosis of the left subclavian artery, which was treated with angioplasty and stent placement, resulting in <10% residual stenosis. Outcome: Following the intervention, a repeat echocardiogram showed only mild calcification of the aortic valve with a mean gradient of 4 mm Hg, significantly lower than the previous findings. Discussion: This case underscores the potential for subclavian artery stenosis to mimic severe AS on echocardiography, particularly when using the Pedhoff probe. It highlights the need for clinicians to consider vascular anomalies in the differential diagnosis when echocardiographic doppler findings do not correlate with clinical symptoms or with the structural apperance of the valves. Conclusion: Comprehensive assessment and consideration of alternative diagnoses are crucial in patients with suspected severe AS, especially when using Doppler echocardiography. Awareness of the potential for subclavian artery stenosis to cause false-positive echocardiographic findings can prevent unnecessary interventions and guide appropriate management.

Abstract 4113301: A Rare Cause of Recurrent Heart Failure Exacerbations After Transcatheter Aortic Valve Replacement: Ventricular Septal Defect and Significant Paravalvular Leak

Introduction: Ventricular septal defects (VSD) have been reported in <1% of cases following transcatheter aortic valve replacement (TAVR). A retrospective record review of VSD cases occurring after all TAVRs performed between January 2012 and September 2020 at one urban US tertiary hospital showed 0.37% incidence of these complications, mostly with small and restrictive VSD. Case Description: An 80-year-old woman with history of hypertension, HFpEF, and TAVR 8 months prior for severe aortic stenosis (AS) at an outside hospital presented with worsening dyspnea and peripheral edema over the past month. Blood pressure was 148/53mmHg, heartrate 83bpm, oxygen saturation 98% and auscultation revealed a harsh holosystolic murmur 4/6 loudest at left sternal border and bibasilar rales. Chest X-ray showed pulmonary edema and bilateral pleural effusions. NT-proBNP was 17,000pg/ml and troponin-I was negative. Echocardiogram showed EF55-60%, a membranous VSD, severe pulmonary hypertension, and moderate paravalvular leak (PVL). Coronary angiogram showed non-obstructive disease. Right heart catheterization confirmed significant left to right shunting. Treatment/Outcomes: Cardiothoracic surgery was consulted for multidisciplinary evaluation, and joint recommendation was for surgical aortic valve replacement and VSD closure which was successfully performed. One month follow-up echocardiogram showed normal aortic valve gradient without PVL and normal right ventricular pressure. Discussion/Conclusion: Numerous potential risks such as severe and uneven calcification of the native valve, an elliptic aortic annulus, excessive valve sizing, or elevated valve placement can lead to VSD. A recurrent element in many of these instances involves direct trauma to the septum caused by the implanted valve, exacerbated by subsequent annulus dilation. The presence of PVL is common post-TAVR and is an established independent prognostic factor, while VSD post-TAVR is uncommon, but may be associated with low implantation position relative to the aortic annulus or post-implantation balloon dilatation. Rarely, worsening VSD may require operative management. In this case, the improvement of heart failure symptoms and echocardiogram parameters after transthoracic surgery confirmed successful repair.

Abstract 4135059: Enhanced Expression of iNOS is Associated with Aortic Valve Stenosis in Patients Undergoing Hemodialysis

Background: Patients undergoing hemodialysis (HD) have a greater prevalence of aortic valve calcification and aortic valve stenosis (AS). High prevalence and rapid progression of AS have been reported in patients undergoing HD. However, the precise mechanism of the relationship between HD treatment and rapid development of AS is still unknown. Several findings suggest a pivotal role for oxidative stress in the progression and acceleration of atherosclerosis in patients undergoing HD. A considerable number of human diseases have an inflammatory component, and a key mediator of immune activation and inflammation is inducible nitric oxide synthase (iNOS). Overexpressed iNOS has been implicated in numerous pathogenetic processes, including coronary plaque destabilization. To elucidate the role of iNOS in the aortic valve of patients with AS, we studied its presence in aortic valve specimens from patients with AS undergoing HD. Methods: We enrolled 32 patients undergoing HD and 42 patients not undergoing HD who had severe AS and underwent AVR. Valve samples were analyzed immunohistochemically with antibodies against macrophages, microvessels, iNOS and 4-hydroxy-2-nonenal (4-HNE), an index of lipid peroxidation. Immunoreactivity was quantified with computer-aided polarimetry. Double immunostaining was also performed to identify cell types that stained positive for iNOS. Results: Quantitative analysis demonstrated that macrophage-, iNOS-, and 4-HNE-positive areas, as well as the number of microvessels, were significantly higher in patients with AS undergoing HD (macrophages, P <0.001; microvessels, P <0.05; 4-HNE, P <0.05; iNOS , P <0.001). iNOS-positive areas were positively correlated with 4-HNE-positive areas (R=0.71, P <0.0001). Double immunostaining for both iNOS and macrophages identified iNOS-positive cells as macrophages. Conclusions: These findings indicate that accumulation of iNOS in macrophages may increase oxidative stress and contribute to the rapid progression of AS in patients undergoing HD.

Abstract 4140136: Aortic Valve Calcification Density Performs Better Compared to Absolute Aortic Valve Calcification in Aortic Stenosis Risk Stratification

Aortic valve calcification (AVC) scores can be used to stratify aortic stenosis (AS) severity. Current practice guidelines recommend use of absolute AVC scores without considering body surface area or aortic annulus size. We investigated whether size variation in aortic annular area should be considered in identifying optimal AVC cutoffs for patients with severe AS. This was a retrospective analysis of AS patients that underwent AVC assessment during 2018-2023. Outcomes were composite of valve intervention or all-cause mortality. AVC density was defined by dividing AVC by echo-derived left ventricular tract area. Receiver operator curve analysis determined optimal AVC density scores. Absolute AVC cutoffs of >1200AU for females and >2000AU for males were used to identify severe AS. The study included 749 patients (mean age 78 years, 46% female). Median CT-annulus area was larger for males compared with females (532mm 2 [Q1, Q3: 479, 582] vs. 413mm 2 [370, 458], p<0.001). There was a strong association of absolute AVC with CT-annulus area for both males and females (Spearman coefficient: 0.87 and 0.83, p<0.01 each) respectively (Figure 1). The optimal AVC density cutoffs for males and females were 427 and 326, respectively (accuracy 76%, sensitivity 83%, specificity 51%). Composite outcomes were better differentiated with AVC density (males: HR 2.48, 95% CI: 1.82-3.37, p<0.001; females: HR 2.37 (1.71-3.28), p<0.001) compared with absolute AVC scores (males: HR 1.81 (1.43-2.30), p<0.001; females: HR 2.11 (1.62-2.74), p<0.001) (Figure 2). AVC is strongly associated with CT-annulus area and risk stratification is improved with the use of AVC density compared with absolute AVC. This may be especially helpful in patients with extremes of annulus area sizes.

Abstract 4138348: Mechanical stress-mediated nuclear envelope damage promotes Aortic Valve Calcification through the ZBP1-RIPK3-NF-κB signaling axis

Methods and Results: We describe a comprehensive characterization of the AVICs nucleus landscape as determined by transmission electron microscopy (TEM) of samples obtained from CAVD patients. Turbulence led to nuclear envelope integrity lose in AVICs cultured in shear stress experiments, with three different fluid conditions [static (ST), laminar stress (LS), and oscillatory stress (OS)], indicated by Western blot and immunofluorescence (IF). Silencing lamin A/C (LMNA) through small interfering RNA (siRNA), accelerated nuclear envelope damage , as indicated by Western blot, qPCR, and immunofluorescence (IF). The formation of Z-DNA and its co-localization with Z-DNA binding protein (ZBP1) was observed due to the nuclear envelope damage by IF. Western blot, qPCR, IHC and IF confirmed Z-DNA-induced inflammation in AVICs through the ZBP1-RIPK3-NF-κB signaling pathway. ZBP1 and RIPK3 knockdown with siRNA markedly reduced the protein level of osteogenic markers alkaline phosphatase (ALP), runt-related transcription factor 2 (RUNX2), and bone morphogenetic protein 2 (BMP2) in VICs. In vivo, aortic valve disease was constructed by direct wire injury (DWI), and we showed that overexpression of LMNA by adeno-associated virus significantly decelerated the progression of aortic valve lesion induced by DWI in mice. Conclusion: Excessive mechanical stress can induce damage to the nuclear envelope of AVICs by causing cytoskeletal remodeling, initiating the formation of Z-DNA, and hastening the calcification process in AVICs and CAVD.

Abstract 4138340: Cav-1/FGF2 Axis Mediates Endothelial-Mesenchymal Transition Promoting Aortic Valve Calcification

Background: Calcific Aortic Valve Disease is a common cardiac condition with no effective medication available; severe cases require surgery. Endothelial-to-mesenchymal transition is a key pathological process, yet the genetic regulatory mechanisms involved remain unclear. Research Questions: Investigating the mechanism of Cav-1/FGF2 axis-mediated endothelial-mesenchymal transition in aortic valve calcification. Methods: Histopathology, immunohistochemical staining, and western blot analysis were performed on 5 non-calcified and 5 calcified aortic valves. Proteomic sequencing was conducted on these samples. Single-cell RNA sequencing on 3 calcified aortic valves focused on valve endothelial and interstitial cells to explore key genes and functions in EndoMT. In vivo and in vitro experiments, along with single-cell multi-omics sequencing, elucidated the role and mechanism of the Cav-1/FGF2 axis in EndoMT using endothelial cell-specific EndoMT knockout mouse models and cell models. Results: Proteomics and single-cell sequencing results indicated that Cav-1 is involved in the Endothelial-to-mesenchymal transition process in Calcific Aortic Valve Disease. Cav-1 plays a crucial role in the intermediate transition of EndoMT, interacting significantly with FGF2. Immunohistochemical staining showed high expression of Cav-1 in calcified aortic valves. In vitro experiments demonstrated that Cav-1 mediates Endothelial-to-mesenchymal transition in valve endothelial cells, promoting aortic valve calcification. Molecular docking analysis revealed potential direct interaction between Cav-1 and FGF2 proteins. Elevated Cav-1 gene expression increases the number of cell surface caveolae, enhancing cell surface area, which promotes FGF2 binding to its receptor and activates the TGF-beta receptor, both of which facilitate EndoMT through the Smad pathway. Conclusion(s): Activation of the Cav-1/FGF2 axis promotes endothelial-mesenchymal transition, thereby promoting aortic valve calcification.

Abstract 4138306: Impact of Early Menopause and Hormonal Replacement Therapy on Aortic Stenosis Progressio

Background: Early menopause is associated with an increased risk of cardiovascular disease and mortality in women. The present study aimed to examine the impact of early menopause and hormonal replacement therapy (HRT) on the progression of aortic stenosis (AS). Methods: Thirty-three postmenopausal women (mean age 65 ± 10 years) with mild or moderate AS prospectively recruited in the PROGRESSA study (NCT01679431) were included in this sub-analysis. All patients underwent multidetector computed tomography and Doppler-echocardiography at least twice during follow-up to assess both anatomic and hemodynamic AS severity, based on aortic valve calcification (AVC), mean pressure gradient (MG), and aortic valve area indexed to body surface area (AVA). Annualized changes in AVC, MG and AVAi were calculated between baseline and the last follow-up. Results: Over a median follow-up of 2 [1-4] years, early menopausal women had a faster progression rate of AVC, MG, and AVAi compared to other women: (100[58-130] vs. 23[2-71] AU/year, p=0.03); (2.37[0.82-3.61] vs. 0.31[0.01-1.78] mmHg/year, p=0.04); and (-0.12[-0.23-0.002] vs. -0.004[-0.07-0.08] cm 2 /m 2 /year, p=0.08) respectively. In multivariate analysis adjusted for age, AS severity at baseline, and comorbidities, early menopause remained significantly associated with faster AVC progression (p=0.003). Moreover, after comprehensive adjustment, women who received HRT (45%) had a slower progression rate of AVC (20[10-42] AU/year vs. 62[2-100], adjusted p=0.04). Conclusion: Early menopause is associated with faster progression of AS, both anatomically and hemodynamically. However, the use of HRT is associated with slower progression of AS. Integrating female-specific risk factors, particularly menopausal history and hormonal therapy status, into the clinical management of AS could enhance patient care.

Abstract 4136289: Aortic Valve Calcium as a Predictor of Chronic Kidney Disease in a Multi-Ethnic Cohort: The MESA Study

Background: Aortic valve calcium (AVC) is associated with an increased risk of cardiovascular disease, non-cardiovascular disease such as dementia, and all-cause mortality. Traditional atherosclerotic cardiovascular disease risk factors are associated with both AVC and chronic kidney disease (CKD), but whether there is an association between AVC and CKD is unknown. Objectives: To ascertain whether AVC quantified by cardiac CT scanning is independently associated with the long-term risk of incident CKD among individuals without a previous history of cardiovascular disease. Methods: We examined 6,346 Multi-Ethnic Study of Atherosclerosis (MESA) participants who underwent cardiac CT scanning at Visit 1 (2000-02) and had an eGFR of ≥ 60 mL/min/1.73 m 2 . AVC was quantified using the Agatston method and categorized as 0, 1-99, and ≥100. Incident CKD was defined as an eGFR < 60 mL/min/1.73 m 2 accompanied with an at least 40% decline in eGFR from baseline, and/or a diagnosis of CKD and indicators of end stage renal disease extracted from hospital records using the International Classification of Disease (ICD) codes. We performed Kaplan-Meier survival curve analyses along with multivariable Cox proportional hazard regression models, adjusted for age, gender, race/ethnicity, highest level of education and traditional cardiovascular risk factors along with coronary artery calcium (CAC), lipoprotein (a) (Lp[a]), and the APOE-ε4 genotype to examine the association between AVC (categorical and log-transformed) and incident CKD. Results: Participants had a mean age 62.2±10.1 years, 53% were women, and AVC >0 was present in 795 (12%) participants. During a median follow-up time of 16.9 years, 982 (15%) participants developed incident CKD. AVC examined as a continuous variable was associated with a significantly increased risk of developing CKD (per log-unit [AVC+1] HR 1.06 [95% CI: 1.02-1.10]; p = 0.005). There was a stepwise increased risk for CKD with higher AVC levels ( Figure ). Similarly, in the multivariable adjusted Cox models, participants with AVC ≥100 had a higher risk of incident CKD, compared with the AVC=0 group (HR 1.48 [95% CI: 1.15-1.89]; p = 0.002). The observed associations remained after further adjusting for CAC score ( p = 0.024), Lp (a) ( p = 0.004), and the APOE-ε4 genotype ( p = 0.004). Conclusions: In a multi-ethnic cohort of participants free of CKD at baseline, AVC was independently associated with a higher risk of incident CKD.

Abstract 4119560: Oxidized Phospholipids and Calcific Aortic Valvular Disease

Introduction: Oxidized phospholipids (OxPL) are carried by apolipoprotein B-100-containing lipoproteins (OxPL-apoB) including lipoprotein(a) [Lp(a)]. Both OxPL-apoB and Lp(a) have been associated with calcific aortic valve disease (CAVD). Aims: We aimed to evaluate the independent associations between OxPL-apoB, Lp(a) and the prevalence, incidence, and progression of CAVD. Methods: OxPL-apoB and Lp(a) were evaluated in the Multi-Ethnic Study of Atherosclerosis (MESA) and a participant-level meta-analysis of four randomized trials of participants with established aortic stenosis (AS). In MESA, the association of OxPL-apoB and Lp(a) with aortic valve calcium (AVC) at baseline and 9.5 years was evaluated using multivariable ordinal regression models. In the meta-analysis, the association between OxPL-apoB and Lp(a) with AS progression (annualized change in peak aortic valve jet velocity (V max )) was evaluated using multivariable linear regression models. Results: In MESA, both OxPL-apoB and Lp(a) were independently associated with prevalent AVC (OR (95% CI) per SD: 1.19 (1.07-1.32) and 1.13 (1.01-1.27), respectively) with a significant interaction between the two (p<0.01). Both OxPL-apoB and Lp(a) were associated with incident AVC at 9.5 years when evaluated independently (interaction p<0.01). The OxPL-apoB*Lp(a) interaction demonstrated higher odds of prevalent and incident AVC for OxPL-apoB with increasing Lp(a) levels. Mediation analyses demonstrated that Lp(a) partially mediated the association between OxPL-apoB and AVC. In a 2x2 analysis, the greatest risk for both prevalent and incident AVC was observed when Lp(a) and OxPL-apoB were both above the 80 th percentile ( Figure ). In the meta-analysis, when analyzed separately, both OxPL-apoB and Lp(a) were independently associated with faster increase in V max , but when evaluated together, only OxPL-apoB remained significant (ß 0.07, 95% CI 0.01-0.12, Figure ). Conclusions: OxPL-apoB is an independent predictor of the presence, incidence and progression of AVC and established AS, particularly in the setting of elevated Lp(a) levels and may represent a novel therapeutic target for CAVD.

Abstract 4135861: Lipoprotein(a) and Its Impact on Left Ventricular Remodeling Over a Decade: The Multi-Ethnic Study of Atherosclerosis

Background: Lipoprotein (a) (Lp[a]) is associated with an increased risk of cardiovascular disease and mortality, as well as heart failure and myocardial fibrosis. However, the link between Lp(a) and cardiac remodeling as a pathway to adverse cardiac outcomes remains unknown. Objectives: This study investigated the relationship between Lp(a) levels and longitudinal changes in the left ventricular (LV) remodeling over a decade among individuals without a previous history of cardiovascular disease. Methods: 2,366 Multi-Ethnic Study of Atherosclerosis (MESA) participants who underwent cardiac MRI at Visit 1 (2000-02) and Visit 5 (2010-12) and had available Lp(a) at baseline were examined. Lp(a) was analyzed as a continuous and a categorical variable based on quartiles (Q1[<7.6 mg/dL], Q2[7.6-16.7 mg/dL], Q3[16.8-38.8 mg/dL], Q4[>38.8 mg/dL]). Multivariable linear regression analysis was used to examine the association of Lp(a) with changes in cardiac MRI measures of LV remodeling ( Table ). Results: Participants had a mean age 60±9 years and 53% were women. Over 10-year follow-up, LV indexed volumes decreased, while LV indexed mass and mass to volume ratio increased across all the Lp(a) quartiles. However, LV ejection fraction only decreased in the third and fourth Lp(a) quartiles. Lp(a) examined as a continuous variable was associated with an increase in LV end-systolic indexed volume (per log-unit Lp[a]; β 0.32 mL/m 2 ; P = 0.01), LV indexed mass (per log-unit Lp[a]; β 0.38 g/m 2 ; P = 0.02), and a decrease in LV ejection fraction (per log-unit Lp[a]; β -0.29 %; P = 0.02) over 10 years after adjusting for sociodemographic and traditional cardiovascular risk factors ( Table ). Similarly, the fourth Lp(a) quartile was associated with an increase in LV end-systolic indexed volume (β 1.07 mL/m 2 ; P = 0.01), LV indexed mass (β 1.17 g/m 2 ; P = 0.02) and a decrease in LV ejection fraction (β -1.01 %; P = 0.01) compared to the first Lp(a) quartile after controlling for risk factors. The observed associations remained significant after further adjusting for aortic valve calcium score at Visit 1 ( Table - Model 3), and baseline coronary artery calcium score and interim myocardial infarction ( Table - Model 4). Conclusions: In a multi-ethnic cohort of participants free of cardiovascular disease at baseline, higher Lp(a) levels were independently associated with an increase in LV end-systolic volume and LV mass as well as a decrease in LV ejection fraction over the span of a decade.

Abstract 4117772: Age-Related Differences in Aortic Valve Calcium Progression and the Risk for Aortic Stenosis: Multi-Ethnic Study of Atherosclerosis

Background: Aortic valve calcium (AVC) is strongly associated with an increased risk for severe aortic stenosis (AS). The prevalence of AVC increases with age affecting 40-50% of individuals ≥80 years. The impact of age on the progression of AVC and its association with incident AS remains unknown. Methods: Our study included 6,810 participants (52.9% women) free of cardiovascular disease between ages 45 and 84 from the Multi-Ethnic Study of Atherosclerosis. AVC was measured using non-contrast cardiac CT at Visit 1. Progression was calculated as the change in AVC divided by years between CT scans with up to 10 years between scans. Long term incident AS was adjudicated using medical chart review and echocardiogram data from Visit 6 with a median follow up of 16 years. Multivariable adjusted 1) linear regression was used to examine AVC progression and 2) multivariable adjusted Cox proportional hazards ratios (HR) were used to examine the association of AVC with incident AS. Results: The prevalence of AVC >0 was 4.9% among participants <65 and 23.2% for those ≥65 years old. Among participants with AVC >0, the median AVC was 34.1 (IQR 13-1,113) for participants <65 versus 69.0 (IQR 23-2,453) for participants ≥65. Participants <65 years and ≥65 years had no significant difference in median annualized AVC progression within the baseline AVC categories of 1-99 (10 versus 12 AU/year, p=0.303) and AVC >100 (50 versus 47 AU/year, p=0.846) ( Figure 1 ). Overall, linear regression models showed older age was associated with greater AVC progression (p=0.001), but after additional adjustment for baseline AVC, the association became non-significant (p=0.134). AVC >0 was associated with significantly increased risk of incident AS for both younger (HR 13.37; 95% CI 5.67-31.52) and older participants (HR 10.59, 95% CI 6.77-16.56). Conclusion: We observed a similar progression of AVC for younger versus older persons after adjusting for baseline AVC burden. Additionally, AVC >0 independently conferred at least a ten-fold higher risk for severe AS among both younger and older participants. These findings demonstrate that the AVC progression is primarily associated with baseline AVC burden and that AVC is a strong marker of risk for severe AS for both younger and older persons.

Challenging clinical management of a patient with Gaucher disease type IIIC homozygous for the D409H mutation, aortic valve calcification and porcelain aorta

Background. Gaucher disease is a rare lysosomal storage disorder caused by glucocerebrosidase enzyme deficiency resulting in the cumulative deposition of glucocerebroside in macrophages, predominantly effecting bone marrow, liver and spleen. Gaucher disease type IIIC is a rare subtype that is characterized by cardiovascular involvement, eye-movement disorders, and late-onset neurological symptoms. Case presentation. We present a 14-year-old adolescent boy diagnosed with Gaucher disease type IIIC at age four with a homozygous D409H mutation who developed severe aortic valve stenosis, extensive aortic calcification and a porcelain aorta despite enzyme replacement treatment since the diagnosis. Despite the challenges during the cardiac surgery, we successfully performed transcatheter aortic valve implantation (TAVI). The patient developed a complete atrioventricular block and required a pacemaker after the TAVI. He experienced further complications during the follow-up. Conclusion. The case presents the challenges in the treatment of cardiovascular complications in patients with Gaucher disease and demonstrates the importance of individualized treatment approaches, as well as the potential advantages and complications of TAVI in difficult situations like this.

Impact of sex-specific thresholds for low flow in assessment of prognosis in concordantly and discordantly graded aortic valve stenosis.

Sex-specific low flow was recently defined as stroke volume index (SVi) ≤40 ml/m² in men and ≤32 ml/m² in women. We tested the prognostic association of these cut-offs in patients with aortic stenosis (AS) with concordantly and discordantly graded AS (CGASEL and DGASEL) based on pressure recovery adjusted aortic valve area (energy loss, EL). Data from 1351 patients with asymptomatic AS, peak jet velocity <4m/s and preserved left ventricular ejection fraction enrolled in the Simvastatin and Ezetimibe in AS study was used. DGASEL was defined as EL <1.0 cm² with mean aortic gradient <40 mmHg, and CGASEL as EL ≥1.0 cm² with mean aortic gradient <40mmHg. Patients were further grouped into normal and low flow. Outcome was combined all-cause death and hospitalization for heart failure. CGASEL with normal/low flow was present in 915/253 patients, and DGASEL with normal/low flow in 57/126 patients. During median 4.3 years follow-up, event-free survival was lower in patients with DGASEL irrespective of flow compared to CGASEL with normal flow (p<0.05). In Cox regression analysis, DGASEL with normal or low flow were both associated with increased risk of all-cause death and hospitalization for heart failure after adjustment for age, sex, heart rate, randomized study treatment, hypertension, aortic valve replacement and aortic valve calcification (p<0.05). No survival difference was found between patients with normal vs. low flow within groups of DGASEL or CGASEL. Identification of low flow by the proposed sex-specific thresholds of SVi needs more prognostic validation before application in clinical practice.

A Review of Biomechanical Studies of Heart Valve Flutter

This paper reviews recent biomechanical studies on heart valve flutter. The function of the heart valves is essential for maintaining effective blood circulation. Heart valve flutter is a kind of small vibration phenomenon like a flag fluttering in the wind, which is related to many factors such as a thrombus, valve calcification, regurgitation, and hemolysis and material fatigue. This vibration phenomenon is particularly prevalent in valve replacement patients. The biomechanical implications of flutter are profound and can lead to micro-trauma of valve tissue, accelerating its degeneration process and increasing the risk of thrombosis. We conducted a systematic review along with a critical appraisal of published studies on heart valve flutter. In this review, we summarize and analyze the existing literature; discuss the detection methods of frequency and amplitude of heart valve flutter, and its potential effects on valve function, such as thrombosis and valve degeneration; and discuss some possible ways to avoid flutter. These findings are important for optimizing valve design, diagnosing diseases, and developing treatment strategies.

Prognostic and therapeutic implications of low aortic valve calcium score in patients with low gradient aortic stenosis.

Low gradient (LG) aortic stenosis (AS) poses a diagnostic challenge. Aortic valve calcium score (AVCS) assessment has emerged as a complementary diagnostic method when echocardiography provides discordant results. However, the diagnostic and prognostic value of AVCS in LGAS has not been thoroughly studied. Our aims were to investigate the prognostic importance of AVCS in LGAS and to assess whether symptomatic patients with LGAS and low AVCS may benefit from aortic valve intervention (AVI). 327 symptomatic patients (78.5±7.3 years, 51% women) with severe AS defined by the aortic valve area who underwent computed tomography for transcatheter aortic valve intervention (TAVI) planning were enrolled. AVCS was measured. AVCS<2000 AU in men and<1200 AU in women was considered low AVCS. 243 patients had high gradient (HG) and 84 had LGAS. Low AVCS was present in 25(10%) of the HG and 34(40%) of the LGAS cases. Over a median follow-up period of 4.9 years, 194 deaths occurred. In multivariate analysis, AVCS was a significant independent predictor of all-cause mortality among HGAS (aHR:2.317; CI:1.104-4.861; p= 0.026), but not among LGAS (aHR:0.848; CI:0.434-1.658; p=0.630) patients. After propensity score matching between patients who underwent AVI and those who were medically treated, AVI (94% TAVI) was a significant and independent predictor of survival among LG AS patients with low AVCS even after adjustment for clinical variables (aHR:0.102, CI:0.028-0.369; p<0.001). The prevalence of low AVCS is much higher in LGAS than in HGAS. In symptomatic severe LGAS low AVCS did not entail a better prognosis. AVI is equally beneficial in LGAS patients with high or low AVCS, similarly to HGAS.

Paired single-cell RNA sequencing and T cell receptor sequencing reveals both pro- and anti-inflammatory roles of T cells in patients with calcific aortic valve disease

Abstract Background Calcified aortic valve disease (CAVD) is a chronic unresolvable inflammatory disorder that affects 25% of adults over 65 years old, leading to aortic stenosis, heart failure, and death [1]. Since the molecular mechanisms of its pathogenesis are not well understood, therapies to treat root causes of the disease remain to be developed, with valve replacement as the major intervention strategy currently. Increasing evidence indicates that substantial T cells infiltrate in the aortic valve during calcification, and clonal expanded CD8+ T cells have been identified in the calcificed aortic valve by bulk T cell receptor (TCR) repertoire sequencing [2]. Purpose To decipher T cell heterogenity and understand the precise mechanisms of T cell subsets involving CAVD progression as a basis for novel therapeutic targets. Methods In Cohort 1, we collected aortic valves from CAVD (n=3) and healthy (n=2) patients for single-cell RNA sequencing (scRNA-seq). In Cohort 2, we performed paired scRNA-seq and TCR sequencing (scTCR-seq) on aortic valves obtained from CAVD (n=3) and healthy (n=3) patients to evaluate both gene expression and clonality. Pathway enrichment analysis and cell-cell interaction analysis elucidated the function of T cells in the calcificied valves. Immunohistochemistry and fluorescence-activated cell sorting were recruited to verify the key findings. Results scRNA-seq demonstrated a conversion from anti-inflammatory regulatory T (Treg) cells towards pro-inflammatory T helper 17 (Th17) cells in the calcificied aortic valve. Paired scRNA-seq and scTCR-seq revealed CAVD-related signaling pathways (osteoclast differentiation, NOD-like receptor signaling pathways, IL-17 signaling pathways) significantly upregulated in highly expanded CD8+ T cells in the calcified valve, with upregulation of pro-inflammatory mediators such as IFNG, CCL4 and CCL5, suggesting pro-inflammation functions in clonally expanded T cells. Moreover, we characterized tissue-resident memory T cells, which resembled similar transcriptome and TCR clonality as exhausted T cells in calcified valves. These cells up-regulated anti-inflammation-related transcriptomes but their cellular fraction decreased in calcified valves, implying anti-inflammatory functions of these tissue-resident memory and exhausted T cells. Conclusion This study elucidates transcriptomic and immune profiling of T cells in the calcified aortic valve, therefore shedding light on the pathophysiological mechanisms underlying aortic valve calcification from the novel perspective of tissue-specific T lymphocytes, providing promising targets for immune checkpoint-based therapeutic interventions.

The mechanical environment regulates the extent of aortic valve calcification and thickness: an ex vivo whole mouse heart study

Abstract Background Aortic stenosis (AS) is a narrowing of the aortic valve opening due to calcification and thickening of the leaflets. Calcifications are mostly located at regions with high mechanical stress and disturbed flow suggesting that mechanical environment is important in the regulation of calcification. However, direct evidence linking valve calcification and mechanical stress is still lacking. Recently, we established an ex vivo calcification model for whole mouse hearts (Miniature Tissue Culture System MTCS) which allows the study of the mechanical environment underlying aortic valve calcification. Purpose Aim of this study was to investigate the role of mechanical stress on aortic valve calcification and thickening using the MTCS. Methods Whole mouse hearts were cultured in the MTCS with 2 distinct configurations: 1) medium flowing from the aorta towards the aortic valve to create a continuous closed aortic valve (n=28) and 2) medium flowing from the left ventricle through the aortic valve towards the aorta to create a continuous open aortic valve (n=17) (Figure1). The hearts were subjected to low (300 ul/min) or high (3000 ul/min) flow speed in the presence of calcifying medium (3mM phosphate buffer). After culture for 1 week, the hearts were isolated and fixed overnight with 4% PFA/PBS. Alizarin red staining was performed to determine calcification presence. Quantifications were performed using Caseviewer software. Results Aortic valves in closed configuration exposed to a high flow speed, exhibited significant increase in calcification compared to aortic valves in the closed configuration exposed to low flow speed or aortic valve cultured in the open position (p&amp;lt;0.01, Figure2A). Of interest, the extent of calcification in the aortic valve in closed position exposed to high flow speed strongly correlated with the diameter of the aortic annulus (R²=0.86, p&amp;lt;0.0001, Figure2B). Conversely, leaflet thickness was significantly lower in the closed aortic valve configuration as compared with the open aortic valves configuration (p&amp;lt;0.05, p&amp;lt;0.001, Figure2C). Conclusion Our study demonstrates the influence of mechanical environment on aortic valve calcification and thickening. The presence of calcification in the closed but not the open aortic valve configuration exposed to high flow suggests that increased pressure promotes calcification. The correlation of the extent of aortic valve calcification with the diameter of the aortic annulus implies that configuration of the valve is an important determinator of calcification, with potential implications for the results of aortic valve repair. The presence of a minimal amount of mechanical stress as experienced by aortic valves in the open position, however, resulted in thicker aortic valves, indicating that the right balance of mechanical stresses is required to maintain normal valve morphology. These findings provide valuable insights into AS pathogenesis, guiding future therapeutic approaches.ex vivo cultured aortic valvesCalcification in ex vivo aortic valves

Targeting an altered sphingolipid profile with consecutive lipotoxicity as therapeutic approach to calcific aortic valve disease

Abstract Background Aortic stenosis due to calcific aortic valve disease (CAVD) is the most common valvular heart disease. In symptomatic patients, without repair the prognosis is poor. Treatment to date is based on surgery or catheter-based interventions. Pharmacological therapy options to prevent the progression of valve calcification are not available. In vascular smooth muscle cells, cellular accumulation of sphingolipids is associated with a proinflammatory response in the sense of lipotoxicity. Chronic inflammatory processes are indeed also essential for aortic valve calcification. Purpose With the subsequent goal of developing innovative treatment approaches for CAVD, we therefore aim to elucidate the unclear role of sphingolipids in the development of CAVD. Methods Human aortic valve and plasma samples of CAVD patients and controls (n=26) were analysed by liquid chromatography–mass spectrometry for lipidomics and proteomics. Human aortic valve interstitial cells (hVICs) were stimulated with ceramides. Calcification was detected by alizarin red staining. mRNA expression of bone-related proteins was determined by qPCR. NF-kB pathway was assessed by proteome profiler. Myriocin and GW4869 were applied to inhibit ceramide biosynthesis. PDTC and MCC950 were used to inhibit NF-kB Pathway and NLRP3 activation. ApoE-/- mice received a gain-of-function PCSK9 adeno-associated virus vector and fed high cholesterol diet for 14 weeks. Myriocin was applied orally over the treatment period to inhibit ceramide de novo synthesis in vivo. Murine echocardiography was used to measure transvalvular velocity and left ventricular function. Calcification degree of murine aortic valve was determined by histological staining. Results A significantly altered sphingolipid profile was observed in aortic valve tissue of CAVD patients (Fig. 1). Ceramide species e.g. Cer (17:1;2O/16:0) were increased. Verifying the biological relevance of these findings in vitro, C2-Cer (d18:1/2:0) enhanced hVIC calcification (Fig. 2). Accordingly, ALP activity and mRNA expression of osteogenic genes RUNX2, ALPL and MSX2 were increased. Furthermore, C2-Cer (d18:1/2:0) enhanced NF-kB p65 phosphorylation and NLRP3 activation, while treatment with both PDTC and MCC950 protected against C2-Cer (d18:1/2:0) induced calcification. Supporting the role of sphingolipid biosynthesis in hVIC calcification, Myriocin and GW4869 both reduced ox-LDL-induced VIC calcification which was reversed by C2-Cer(d18:1/2:0). Finally, Myriocin reduced the degree of calcification and transvalvular jet velocity of aortic valve in the PCSK9-AAV ApoE-/- mice model. Conclusion CAVD is accompanied by an accumulation of ceramides causing lipotoxicity in human aortic valve tissue. Pharmacological modulation of sphingolipid metabolism is an effective approach to prevent the development and progression of aortic valve calcification in vivo and should be considered as a future therapeutic strategy in CAVD patients.Volcano Plot of CAVD tissue lipidomicsC2 Ceramide enhances hVIC calcification