Miller, Jordan D.

BACKGROUND: Pharmacological treatments for fibrocalcific aortic valve stenosis (FCAVS) have been elusive for >50 years. Here, we tested the hypothesis that reactivation of oxidized sGC (soluble guanylate cyclase), the primary receptor for nitric oxide, with ataciguat is a safe and efficacious strategy to slow progression of FCAVS. METHODS: We used quantitative real-time reverse transcription polymerase chain reaction, Western blotting, and immunohistochemistry to characterize sGC signaling and the biological effects of ataciguat on signaling cascades related to nitric oxide, calcification, and fibrosis in excised human aortic valve tissue, aortic valve interstitial cells, and mouse aortic valves. We then conducted randomized, placebo-controlled phase I (14-day safety/tolerance) and phase II (6-month efficacy) trials in patients with moderate aortic valve stenosis. RESULTS: In excised human tissue, we found robust losses in sGC signaling despite upregulation of sGC subunits. In vitro, ataciguat increased sGC signaling and reduced BMP2 (bone morphogenetic protein 2) signaling in aortic valve interstitial cells. In mice with established FCAVS, treatment with ataciguat attenuated BMP signaling and slowed progression of valve calcification and dysfunction. In a phase I, randomized, placebo-controlled trial, treatment with ataciguat for 2 weeks was safe and well tolerated in patients with moderate FCAVS ( https://www.clinicaltrials.gov ; Unique identifier: NCT02049203). In a separate phase II, randomized, placebo-controlled trial, treatment with ataciguat for 6 months slowed the progression of aortic valve calcification and tended to slow the progression of valvular and ventricular dysfunction in patients with moderate FCAVS ( https://www.clinicaltrials.gov ; Unique identifier: NCT02481258). CONCLUSIONS: Collectively, this study highlights the therapeutic potential of the targeted restoration of the diseased/inactive form of sGC for treatment of FCAVS. REGISTRATION: URL: https://www.clinicaltrials.gov ; Unique identifier: NCT02049203. URL: https://www.clinicaltrials.gov ; Unique identifier: NCT02481258.

Background: CDCP1 has been associated with reverse remodeling in human dilated cardiomyopathy (DCM) mediating its effect by reducing cardiac fibrosis which has been demonstrated in-vitro using human cardiac fibroblasts (CF) and in-vivo histologically in mice. However, the role of and the molecular mechanisms by which CDCP1 attenuates cardiac fibrosis in-vivo is unknown. Methods: To characterize the transcriptomic profiles of CDCP1 in cardiac fibrosis, Cdcp1 KO FVB/NJ mice were generated, and implanted with osmotic minipumps containing angiotensin II and phenylephrine (Ang II/PE) or saline at age of 10 weeks. There were 4 experimental groups (6 mice each), Saline_WT, Saline_KO, AngII/PE_WT, and AngII/PE_KO. After 4 weeks of AngII/PE induction, mice were euthanized, RNA was extracted from their heart tissue followed by RNA-seq to explore transcriptomic profiles. Fibrosis was histologically determined using picosirius (PSR) staining and was quantified by percentage of fibrosis area. Results: Histological analysis demonstrated that Cdcp1 KO attenuated severe cardiac fibrosis by 32.7% determined by quantification of percentage of fibrosis area. When comparing the transcriptome-wide gene expression in WT mice hearts from Saline to AngII/PE induction, a total of 316 differentially expressed genes (DEGs) were identified ( Fig. 1A ). Expression of Nppb (encodes ProBNP), a marker for heart failure, was significantly upregulated. The most significantly upregulated DEG after AngII/PE induction was Crlf1 ( Fig. 1A ), a gene predominantly expressed in CF, consistent with previous findings. We next compared transcriptomic profiles of AngII/PE induction WT and Cdcp1 KO mice hearts, to explore the role of CDCP1 in cardiac fibrosis. A total of 163 DEGs were identified ( Fig. 1B ). Notably, Cdcp1 -mediated DEGs enriched GO Pathways associated with extracellular matrix organization (Biological Process) and collagen-containing extracellular matrix (Cellular Component) ( Figs. 1C, 1D ), pivotal processes in cardiac fibrosis. In addition, Cdcp1 KO resulted in several “top” DEGs (including Rnase1, Ccl7, Ccl12 Il21r , and Il6 ) which have known function in inflammation, suggesting that CDCP1 might attenuate cardiac fibrosis through immune regulation . Conclusion: Our findings underscore the pivotal role of CDCP1 in modulating cardiac fibrosis in-vivo, potentially through immune regulation. Targeting CDCP1 may offer promising therapeutic avenues for mitigating myocardial fibrosis.

Abstract Background Multiple sclerosis (MS) is an immune-mediated central nervous system (CNS) demyelinating disease which is diagnosed based on CNS lesions' spatial and temporal dissemination. The 2017 revised McDonald Criteria include cerebrospinal fluid (CSF) specific oligoclonal bands (OCBs) to indicate dissemination in time, but their assessment is labor-intensive and requires a paired serum sample. Kappa immunoglobulin free light chain in CSF (KCSF) has emerged as a more sensitive but less specific MS biomarker, measured from a single CSF sample. Our institution's MS testing panel uses KCSF initially to rule out MS, with reflex to OCB for positive confirmation if KCSF is borderline positive (≥0.06 mg/dL). However, manual correlation of laboratory and clinical data hinders the direct comparison of the diagnostic performance between these markers. Herein we report a retrospective assessment of KCSF and OCB diagnostic performance using automated chart review. Methods Between January 1st, 2021 and December 31st, 2022, there were 1,237 patients with simultaneously measured OCB and KCSF. CSF-specific OCBs were measured by separating CSF and serum proteins using an agarose isoelectric focusing gel with a pH gradient from 3-10 (Sebia). After transfer to nitrocellulose paper, antisera were applied to visualize IgG specific bands and quantify CSF-specific OCBs. KCSF was quantitated using nephelometry by applying free light chain antisera (Binding Site) to CSF samples using a Siemens BNII analyzer. An automated chart review process utilizing keyword matching was implemented to identify patients with MS. An initial filter phase required presence of the term “multiple sclerosis”, while a second required the presence of any of the following terms: “primary progressive,” “secondary progressive,” “relapsing remitting,” “progressive relapsing,” or “tumefactive” to identify patients with diagnosed MS. The automated method’s performance was then compared to a manual chart review of 200 randomly selected patients. Subsequently, the automated process was used to determine the MS diagnosis status of all 1,237 patients. Results Of the 200 manually reviewed patients, 44 MS diagnoses were found. The automated process correctly identified 41 of these with a resulting sensitivity and specificity of 93.18% and 95.51%, respectively. Applied to the entire cohort, the automated review identified 275 MS diagnoses. For the entire cohort, the area under the curve (AUC) for the receiver operating characteristic (ROC) curves for OCB and KCSF were 0.824 (95% confidence interval: 0.794, 0.854) and 0.808 (0.777, 0.839) respectively. Our institution’s diagnostic cutoff of ≥2 for OCB yielded a sensitivity and specificity of 74.2% (68.3%, 79.1%) and 84.9% (73.8%, 89.5%), respectively. The best combination for sensitivity and specificity for KCSF with the Youden index yielded a cutoff ≥0.0654 mg/dL (sensitivity of 79.6% [73.1%, 84.0%] and specificity of 72.4% [62.8%, 76.8%]), which is similar to previous publications. This supports the use of automated chart review and suggests the KCSF cutoff has been stable over time. Conclusions KCSF exhibits comparable MS diagnostic performance to OCB yet only requires one CSF sample and can be measured rapidly. When validated, mining unstructured data allows for test performance characterization in large patient cohorts without the need for manual chart review.

Objective: We recently discovered that activation of oxidized soluble guanylate cyclase with ataciguat (ATA) slows valve calcification and dysfunction in mice and humans with moderate aortic valve stenosis (AVS). We examined if ATA would attenuate multiple mechanisms contributing to AVS and end organ dysfunction associated with AVS, and hypothesized: 1) ATA attenuates osteogenic signaling in valve tissue from mice with AVS, thereby attenuating calcification and valve dysfunction, 2) AVS increases rates of sudden cardiac death in our model, and 3) treatment with ATA prevents QT prolongation and deleterious electrical remodeling commonly observed in AVS. Methods: Functional, molecular, and histopathological changes in valve tissue along with QT interval duration were measured in 3 independent groups of mice: non-AVS, AVS, and AVS+ATA (WD for 6 months then WD+ATA for 3 months (AVS+ATA). We gave 5 mg/kg isoproterenol (ISO, i.p.) to ldlr -/- /ApoB100-only mice fed normal chow (non-AVS) or western diet (AVS, fed WD for 9 months) to assess risk for malignant arrhythmias. Retrospective analysis of QT interval was also done on data from our recent Phase 1 clinical trial. Results: AVS+ATA mice had less valve calcification and slower progression of valve dysfunction through attenuation of canonical BMP signaling and reductions in Runx2 and SPP1 compared to AVS mice. QT prolongation was evident in AVS mice, and was significantly attenuated by treatment with ATA (p= 0.001) and coarsely associated with severity of valvular or ventricular function. While all non-AVS mice survived for 24 hours after ISO, 40% of mice with AVS reached a moribund equivalent within 4 hours. Retrospective analyses also showed reductions in QT interval duration in patients treated with 200 mg/day ATA for 14 days (p < 0.05). Conclusion: Collectively, this is the first study reporting increased propensity for malignant arrhythmias in our mouse model of AVS, QT prolongation as a likely underlying mechanism, and restoration of cardiac conduction dynamics with targeted restoration of nitric oxide signaling. Future studies focused on the role of sGC in the evolution and prevention of AVS and consequent deleterious electrical remodeling/malignant arrhythmia risk is warranted.

Thoracic aortic aneurysm (TAA) is a prominent condition that can progress to both aortic dissection and rupture, and has a higher prevalence in patients with bicuspid aortic valves. While smooth muscle cell proliferation and matrix remodeling are hallmarks of TAA, mechanisms regulating these phenomena are poorly understood. Thus, we tested the hypothesis that mTOR signaling, a positive regulator of cell proliferation and matrix remodeling enzyme elaboration, is increased in human TAA samples from patients with tricuspid and bicuspid aortic valves. Tissue was acquired at the time of operation, and mTOR activation was measured in aortic tissue from patients with TAA and tricuspid aortic valves (n = 29), TAA and bicuspid aortic valves (n = 29) and normal, non-aneurysmal aortic tissue from organ donors (n = 13). qRT-PCR expression of mTOR (1.29±0.1), Raptor (1.31±0.09, an mTORC1-specific subunit), and Rictor (1.44±0.1, an mTORC2-specific subunit) was significantly increased in tricuspid TAA samples compared to normal aorta (1 ±0.1; p < 0.05 for all). Similar changes were observed in patients with bicuspid TAA, as expression of mTOR (1.33±0.04), Raptor (1.27±0.06) and Rictor (1.65±0.09) were increased compared to normal (p < 0.05 for all). Importantly, protein levels of p-S6K and p-4E-BP1, both of which are well-defined targets of mTORC1 signaling, were increased by approximately two-fold in bicuspid and tricuspid TAA samples compared to normal aorta. Collectively, to our knowledge, this is the first report suggesting increased activity of both mTORC1 and mTORC2 signaling in human thoracic aortic aneurysm. We propose that inhibition of mTOR signaling may be a useful and efficacious intervention to inhibit cellular proliferation and matrix metalloproteinase activation in TAA, ultimately slowing aneurysmal dilatation and progression to aortic dissection and rupture.

Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthases (NOS), and is degraded primarily by dimethylarginine dimethylaminohydrolase-1 (DDAH1). While ADMA is elevated in patients with fibrocalcific aortic valve stenosis (FCAVD), is it unknown whether tissue levels of DDAH1 are altered in FCAVD, and whether systemic elevations in ADMA levels accelerate initiation and/or progression of FCAVD. In the present study, we measured expression of DDAH1 in normal and stenotic human aortic valves, and tested the hypothesis that increases in ADMA worsen hypertension and endothelial dysfunction and accelerate the initiation and progression of FCAVD in hypercholesterolemic mice. In tissue from humans with FCAVD, expression of DDAH1 was significantly increased compared to normal valve tissue (normal = 1.0 ± 0.1, FCAVD = 1.3 ± 0.1, ⋔⋔Ct method; p < 0.05). To experimentally alter DDAH1 levels, we used low density lipoprotein receptor-deficient, apolipoprotein B100-only (LA) mice that were either wild-type (D +/+ ) or heterozygous (D +/- ) for DDAH1. Blood pressure (tail cuff), aortic endothelial function (isolated organ bath), and aortic valve function (echocardiography), were measured at 3 and 6 month time points in these mice. Compared to LA-D +/+ mice, systolic arterial pressure was significantly increased in LA-D +/- mice at both 3 month (121±4 mmHg and 144±4 mmHg) and 6 months (138±6 mmHg and 148±4 mmHg). Aortic endothelial function progressively declined with prolonged hypercholesterolemia, but maximum relaxation to acetylcholine did not differ between LA-D +/+ and LA-D +/- mice at 3 or 6 months. While aortic valve function was similar between LA-D +/+ and LA-D +/- mice at 3 months (peak transvalvular velocity: 1.98±0.07 m/sec versus 1.94±0.06 m/s, respectively), aortic valve function was significantly worse in LA-D +/- (2.41±0.11 m/sec) compared to LA-D +/+ mice (2.20±0.09 m/sec) at 6 months (p < 0.05). In conclusion, our data support a working model in which increases in valvular DDAH1 levels protect against progression of FCAVD. Importantly, loss of DDAH1 in mice does not alter aortic valve function during early stages of FCAVD, but accelerates progression of FCAVD independently of changes in endothelial function.

Background: We have previously demonstrated by performing a genome-wide association study that a genetic locus resulting in ↓CDCP1 expression was associated with myocardial recovery in patients with dilated cardiomyopathy (DCM). We have subsequently shown that ↓CDCP1 decreases human cardiac fibroblast proliferation in vitro , however, its potential role in modulating cardiac fibrosis and function in vivo is unknown. Methods: A Cdcp1 knock-out (KO) mouse was generated on FVB/NJ background. Angiotensin II/Phenylephrine (AngII/PE) or saline was infused by an osmotic pump into mice ( Cdcp1 KO n=20; wild type/wt n=18) for 4 weeks. Echocardiography was performed at 0 and 4 weeks. Picrosirius red (PSR) staining was performed on cross-sectional heart tissue to identify cardiac fibrosis, which was quantified, using ImageJ software, as a percentage of left ventricular (LV) area stained by PSR. Results: LV mass index (LVMI) significantly increased in AngII/PE wt mice as compared to saline wt (figure a. 3.65±0.15 mg/g vs 3.05±0.17mg/g, p=0.01), however, AngII/PE Cdcp1 KO mice had significantly decreased LVMI as compared to AngII/PE wt mice (figure a. 3.17±0.09 mg/g vs 3.65±0.15 mg/g, p=0.01). In addition, fractional shortening trended higher in AngII/PE Cdcp1 KO mice as compared to AngII/PE wt mice (figure b. 50.36±2.06% vs 45.48±1.43%, p=0.07). Extent of cardiac fibrosis was significantly higher in AngII/PE wt mice as compared to saline wt (figure c. 15.79±0.49% vs 7.93±0.39%, p<0.0001), however AngII/PE Cdcp1 KO mice had significantly reduced cardiac fibrosis as compared to AngII/PE wt mice (figure c. 9.41±0.43%, p<0.0001). Conclusions: CDCP1 KO significantly reduces pressure over-load induced LV hypertrophy and cardiac fibrosis. Its role as a novel therapeutic target in attenuating fibrosis and improving cardiac function in DCM needs to be further explored.

In Marfan syndrome, mutations in the fibrillin gene are known to result in aortic dilatation and aneurysm formation, which is associated with impaired endothelial function and increased vascular stiffness. Previous studies suggested these phenotypes may be partly attributable to overactivation of TGF β signaling, which leads to increased reactive oxygen species production, extracellular matrix production, and increased matrix metalloproteinase activity in aortic wall. To our knowledge, however, there are no studies comprehensively evaluating the relationships between conduit vessel vasomotor function, vascular stiffness, and aortic dilatation in young and aged models of Marfan syndrome. Thus, the aim of this study was to test the hypothesis that carotid arterial vasomotor dysfunction and increased vascular stiffness precede aortic dilatation in fibrillin mutant mice. We used wild‐type (+/+) or fibrillin mutant (+/C1037G) mice fed a normal chow diet for 3 months (young) or 14 months (old), measured changes in vasomotor function and vascular stiffness in carotid arterial segments using pressurized arteriography, aortic dimensions using high resolution echocardiography, gene expression using qRT‐PCR, and protein levels using immunohistochemistry. Consistent with our hypothesis, young +/C1037G mice had impaired endothelium‐dependent relaxation to acetylcholine (ACH) compared to +/+ littermates, but did not exhibit changes in vascular stiffness. In contrast, old +/C1037G mice did not have significant changes in vasomotor responses to ACH compared to +/+ mice, but displayed markedly increased vascular stiffness compared to their wild‐type littermates. Aortic dimensions were significantly increased in young +/C1037G mice compared to their +/+ littermates, and these changes were exaggerated further with aging. Consistent with previous reports, we found increased TGF beta‐1 expression and canonical TGF beta signaling (p‐Smad2/3 levels) in +/C1037G mice. Interestingly, we also observed increased expression of Runx2 with aging in +/+ mice with aging, and expression of Runx2 was substantially increased in +/C1037G mice compared to their +/+ littermates at both time points. Collectively, our data show that vasomotor function and vascular stiffness in carotid arterial segments change independent of changes in aortic root diameter, suggesting significant heterogeneity in the mechanical and molecular responses to fibrillin deficiency throughout the vascular system. Support or Funding Information NHLBI (R01‐111121)

The structural protein, fibrillin, is important for maintaining the strength and reliability of connective tissue found in blood vessels and heart valves. Mutations in fibrillin‐1 result in Marfan syndrome and leads to left‐sided heart valve regurgitation, aortic root dilatation and aortic aneurysm. While previous studies have focused predominantly on young fibrillin‐mutant mice, hemodynamically significant disease generally does not emerge in patients with fibrillin mutations until later in life. Thus, this study aims to evaluate molecular and functional changes in heart valves from aged fibrillin mutant mice. Wild‐type (+/+) or fibrillin mutant (+/C1037G) mice were fed normal chow diet for 3 months (young) or 14 months (old). Heart valve function, aortic dimensions, and cardiac function were evaluated using high‐resolution echocardiography, and gene expression was measured using qRT‐PCR. Consistent with previous reports, we found no evidence of mitral regurgitation in young mice of either genotype. In old +/C1037G mice, however, we did find evidence of mitral regurgitation (~5%). In contrast, aortic valve regurgitation was evident in a relatively large proportion of +/C1037G mice at both ages (~20% at both time points), and was not detected in any +/+ animals. Interestingly, left ventricular dimensions were not associated with the presence of mitral regurgitation, but aortic root dilatation was present in all mice with aortic valve regurgitation. To gain insight into the molecular underpinnings of these phenotypic changes, we measured expression of IL‐10 (an anti‐inflammatory gene) and Runx2 (a pro‐proliferative gene that drives matrix remodeling), both of which are genes previously implicated in the pathogenesis of mitral and aortic valve disease. In young animals, expression of IL‐10 in mitral valves tended to be increased in +/C1037G mice compared to +/+ littermates. With aging, IL‐10 increased substantially in mitral valves from in +/+ mice, and increased further in +/C1037G littermates. Similar trends were observed in expression of Runx2 in mitral valves from both genotypes of mice. While IL‐10 followed a similar pattern in aortic valve tissue from young and old mice of both genotypes, Runx2 was not significantly increased across any of the groups. Collectively, while our data suggest that there are substantial, potentially adaptive molecular changes that occur during development of regurgitant valvular diseases, aortic dilatation may be a key early factor driving development of aortic regurgitation. Future experimental work aimed at determining the relative mechanical and molecular contributions to development of heart valve regurgitation will be critical to development of novel therapeutic strategies in patients with syndromes secondary to fibrillin mutations. Support or Funding Information NHLBI (R01‐HL111121)