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Sirtuin-6 (SIRT6) is a protein deacetylase that functions in several pathways related to aging, as complete loss of SIRT6 in mice elicits multiple age-associated phenotypes and premature death. While increasing age is a major risk factor for development of calcific aortic valve disease (CAVD), it is unclear whether loss of SIRT6 accelerates aortic valve calcification. Here, we report the role of SIRT6 in a subset of aortic valve interstitial cell progenitors (neural crest-derived VICs) by crossing mice carrying Cre recombinase under the control of a PAX3 promoter with mice harboring a SIRT6 gene that can be conditionally inactivated (SIRT6 FL/+ ). We hypothesized that tissue-specific reductions of SIRT6 would increase calcification, increase pro-osteogenic signaling, and impair aortic valve function. We examined mice of the genotypes Cre-/SIRT6 FL/+ or Cre+/ SIRT6 FL/+ at two different time points: young (3 months) and old (14-24 months). First, we used qRT-PCR to demonstrate that SIRT6 was effectively reduced in the aortic valve from PAX3-cre/SIRT6 FL/FL mice. Although reductions in SIRT6 did not change aortic valve calcium (Alizarin Red) in young mice, reducing SIRT6 increased valvular calcium levels in old mice compared to their WT littermates. Interestingly, protein levels of osterix were substantially increased in young, SIRT6-deficient mice compared to WT littermates but were paradoxically decreased in old SIRT6-deficient mice. Regardless of age, histopathological, or molecular changes, we did not observe detectable changes in cusp separation distance at any time point. Collectively, these data suggest that, contrary to our hypothesis, depletion of SIRT6 in neural crest cells—which are key progenitors of valve interstitial cells—is not sufficient to impair aortic valve function. Importantly, this finding suggests that age-associated reductions in SIRT6 may be a permissive context for development of CAVD but relies heavily on the presence of additional risk factors.

Key Points Activin A is implicated in profibrotic and prosenescent kidney injury and correlates with kidney injury markers in animals and humans. Follistatin, through activin A antagonism, reduces senescence burden, macrophage infiltration, and proinflammatory pathway activation in murine diabetic kidney disease. Follistatin and other antagonists of activin A signaling pathways may be promising, novel therapeutics for diabetic kidney disease. Background Circulating activin A, an inflammatory mediator implicated in profibrotic kidney injury and cellular senescence-induced adipose tissue dysfunction, is increased in human diabetic kidney disease (DKD) and directly correlates with kidney dysfunction. We tested the hypothesis that activin A increases kidney injury, senescent cell abundance, and macrophage infiltration in DKD and antagonism through follistatin (FS) therapy diminishes these effects. Methods An accelerated nephropathy type 2 diabetes (db/db) mouse model was generated by implantation of angiotensin II-loaded osmotic minipumps resulting in increased albuminuria and glomerular and tubular injury. Kidney repair effects of FS (5 µ g intraperitoneal; two doses) were assessed through markers of kidney injury, fibrosis, inflammation, cellular senescence, and macrophage infiltration. In vitro studies examined antiactivin effects of FS on high glucose-exposed human monocytes, renal fibroblasts, and renal tubule epithelial cells. Results Activin A antagonism with FS reduced senescence (p19), proinflammatory (including senescence-associated secretory phenotype), and profibrotic markers including activin A. FS improved kidney morphology, restored podocyte markers (nephrin and Wilms tumor-1), and reduced kidney injury biomarkers, albuminuria and kidney fibrosis. FS decreased kidney macrophage and leukocyte infiltration and absent in melanoma 2 inflammasome activation. FS seemed to suppress inflammation through the toll-like receptor-4/NF kappa-light-chain-enhancer of activated B cells pathway in vivo further supported in human macrophages in vitro . In addition, FS reduced hyperglycemia-induced renal fibroblast activation and renal tubule epithelial cell senescence in vitro . Conclusions Activin A is a mediator of kidney injury through macrophage-associated inflammation in murine DKD. FS acts through senomorphic activities which inhibit profibrotic, proinflammatory, and prosenescence signaling by activin A. Hence, antiactivin targeting may aid in the development of a promising, novel therapeutic for DKD.

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.

Introduction: Bispecific antibodies (BsAbs) epcoritamab (Epco) and glofitamab (Glofit) are approved in the US for the treatment of patients (pts) with relapsed or refractory (r/r) large B-cell lymphomas (LBCLs) based upon high response rates and manageable toxicity profiles reported in pivotal trials. However, data on outcomes for pts treated with BsAbs outside of clinical trials are limited. As such, we sought to investigate the safety and efficacy of Epco and Glofit for the treatment of pts with r/r LBCLs in real-world settings. Methods: We performed a retrospective study including 19 US centers evaluating pts with r/r LBCLs receiving commercially available Epco or Glofit between 2023 and 2024. Baseline characteristics and outcomes were extracted from medical records. Efficacy outcomes included overall response rate (ORR), complete and partial response (CR, PR) rates, progression-free survival (PFS) and overall survival (OS), analyzed using the Kaplan Meier method. Key safety outcomes included cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS). Results: As of May 31, 2024, a total of 209 pts with r/r LBCLs were treated with Epco (n=139) or Glofit (n=70). Median age at BsAb start was 67 years (interquartile range [IQR] 58-76). Most were male (62.2%), and the most common histology was diffuse large B-cell lymphoma (n=155 [74.2%]). Of pts with molecular testing (n=168), 34 (20.2%) had double/triple hit lymphoma. Most (78.9%) had Ann Arbor stage III-IV disease and 91% had an ECOG performance status of ≤1. Median number of lines of treatment before BsAb was 3 (range 1-12). Of pts with known response assessment following front-line therapy, 54 (32.1%) had primary refractory disease, defined as failure to achieve PR or CR to front-line therapy. Most pts (n=126 [60.3%]) received prior chimeric antigen receptor (CAR) T-cell therapy and 21 (10.0%) received prior autologous hematopoietic progenitor cell transplant. CD20 expression was assessed in 106 biopsies (50.7%) prior to BsAb and was positive in 90 (84.9%). A total of 172 pts were response-evaluable. The best ORR was 50.6%, with CR in 41 (23.8%), PR in 46 (26.7%), stable disease in 10 (5.8%), and progressive disease in 75 (43.6%). The ORR and CR rates were 49% and 23% for Epco, and 53% and 25% for Glofit, respectively. With a median follow-up of 5 months, the median PFS was 2.7 months (95% CI 2.0, 3.9) and median OS was 7.2 months (95% CI 6.1, not reached [NR]). The estimated PFS and OS at six months were 35% (95% CI 29%-43%) and 59% (95% CI 52%-67%), respectively. For pts who relapsed after CAR T-cell therapy, the median PFS and OS were 2.5 (95% CI 1.8, 4.7) and 7.8 (95% CI 6.2, NR) months, respectively. Pts without detectable CD20 expression via immunohistochemistry or flow cytometry prior to BsAb therapy had significantly inferior outcomes (PFS 1.1 vs 3.4 months, P < 0.001 and OS 1.3 vs 13 months, P < 0.001) compared to pts with detectable CD20. CRS of any grade occurred in 82 pts (39.2%), corresponding to 51.1% and 28.6% of pts treated with Epco and Glofit, respectively. Grade ≥3 CRS occurred in 9 (4.3%), all of whom received Epco. CRS was treated with tocilizumab in 43 pts (53.1%). ICANS occurred in 24 (11.5%) pts; any grade ICANS occurred in 13.7% and 7.1% of pts treated with Epco and Glofit, respectively. Grade ≥3 ICANS occurred in 6 pts (2.9%), 5 of which received Epco (3.6%). Of those experiencing ICANS, 66.0% were treated with corticosteroids. Two pts had deaths attributed to ICANS and one to CRS. A total of 122 (58.4%) pts discontinued therapy after a median of 3 cycles (range 0-48), 91 (74.6%) due to disease progression and 31 (25.4%) due to adverse events (15 due to infection, 3 due to ICANS, 2 due to CRS, and 11 for other reasons). Conclusions: In this heavily pretreated group with adverse disease features and high rate of CAR T exposure, the response rates, PFS, and OS following BsAb therapy were lower compared to pivotal trials. Poor outcomes were observed for those without detectable CD20. Rates of ICANS and grade ≥3 CRS were higher than reported in pivotal trials, as was the rate of discontinuation of therapy due to adverse events. Future efforts to improve outcomes may include intensified dose escalation protocols to more rapidly achieve disease control, combination therapies, and characterization of clinicopathologic factors that predict response to BsAb to identify subgroups of pts who can derive maximal benefit from these agents.

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.