Specific Detection of Prostate Cancer Cells in Urine by RNA in Situ Hybridization

The Journal of urology

2021 Feb 22

Eskra, JN;Rabizadeh, D;Zhang, J;Isaacs, WB;Luo, J;Pavlovich, CP;
PMID: 33617332 | DOI: 10.1097/JU.0000000000001691

Noninvasive tests that can accurately detect prostate cancer are urgently needed for prostate cancer diagnosis, surveillance, and prognosis. Exfoliated prostate cells captured in urine represent a promising resource for noninvasive detection of prostate cancer. We investigated performance of a novel cell-based urine test for detection of clinically significant prostate cancer. We previously developed a multiplex RNA in situ hybridization (RISH) assay targeting NKX3-1, PRAC1 and PCA3 that enables identification and quantification of malignant and benign prostate cells released into urine. We investigated application of the assay for prostate cancer detection in a cohort of 98 patients suspected of harboring prostate cancer. Urine was collected following digital rectal exam and the sediment was isolated and evaluated by RISH. Samples were scored based on cellular expression of RISH targets. Cells of prostate origin were defined by positivity for NKX3-1 and/or PRAC1, and prostate cancer cells by positivity for PCA3. Prostate cells (NKX3-1/PRAC1+ cells) were detected in 69 samples, among which 20 were positive for PCA3 (i.e., positive for prostate cancer cells). Comparison of RISH results with biopsy outcome and clinical variables revealed that positivity for cancer by RISH significantly correlated with intermediate/high risk cancer (p=0.003), PSA density (p=0.022), significant disease (p <0.0001), and Gleason score (p=0.003). The test was 95% specific and 51% sensitive for detection of clinically significant prostate cancer. Identification of exfoliated prostate cancer cells in urine by RISH provides a novel tool for highly specific and noninvasive detection of prostate cancer.

A Murine Calvarial Defect Model for the Investigation of the Osteogenic Potential of Newborn Umbilical Cord Mesenchymal Stem Cells in Bone Regeneration

Plastic and reconstructive surgery

2023 May 24

Stanton, E;Feng, J;Kondra, K;Sanchez, J;Jimenez, C;Brown, KS;Skiles, ML;Urata, MM;Chai, Y;Hammoudeh, JA;
PMID: 37224290 | DOI: 10.1097/PRS.0000000000010754

The standard graft material for alveolar cleft repair (ACR) is autogenous iliac crest. However, a promising alternative potential graft adjunct - newborn human umbilical cord mesenchymal stem cells (h-UCMSC) - has yet to be explored in vivo. Their capacity for self-renewal, multipotent differentiation, and proliferation allows h-UCMSC to be harnessed for regenerative medicine. Our study seeks to evaluate the efficacy of using tissue-derived h-UCMSC and their osteogenic capabilities in a murine model to improve ACR.Foxn1 mice were separated into three groups with the following calvarial defects: (1) no-treatment (empty defect; n=6), (2) poly (D,L-lactide-co-glycolide) (PLGA) scaffold (n=6), and (3) h-UCMSC with PLGA (n=4). Bilateral 2-mm diameter parietal bone critical-sized defects were created using a dental drill. Micro-CT imaging occurred at 1, 2, 3, and 4 weeks postoperatively. The mice were euthanized 4 weeks postoperatively for RNAscope analysis, immunohistochemistry, and histology.No mice experienced complications during the follow-up period. Micro-CT and histology demonstrated that the no-treatment (1) and PLGA-only (2) defects remained patent without significant defect size differences across groups. In contrast, the h-UCMSC with PLGA group (3) had significantly greater bone fill on micro-CT and histology.We demonstrate a successful calvarial defect model for the investigation of h-UCMSC-mediated osteogenesis and bone repair. Furthermore, evidence reveals that PLGA alone has neither short-term effects on bone formation nor any unwanted side effects, making it an attractive scaffold. Further investigation using h-UCMSC with PLGA in larger animals is warranted to advance future translation to patients requiring ACR.Our results demonstrate a successful murine calvarial defect model for the investigation of h-UCMSC-mediated osteogenesis and bone repair and provide preliminary evidence for the safe and efficacious use of this graft adjunct in alveolar cleft repair.

Selective Inhibition of Rho Kinase 2 Limits No Re-Flow in Rat Hearts Following Ischemia/Reperfusion

Circulation

2022 Jan 01

Pearce, L;He, D;Davidson, SM;Yellon, DM;
| DOI: 10.1161/circ.146.suppl_1.11926

Introduction: Myocardial infarction causes several types of injury to the myocardium including lethal cell injury and ‘no-reflow’ (NRF) /microvascular obstruction (MVO). Nonselective Rho Kinase (ROCK1/2) inhibitors such as Fasudil, ameliorate myocardial ischemia/reperfusion (I/R) injury but cause unwanted hypotension. Selective ROCK2 inhibitors (e.g.: KD025) are safe in clinical trials without causing haemodynamic compromise, however they have not been investigated in myocardial I/R. ROCK inhibitors prevent vascular smooth muscle cell (VSMC) contraction; such VSMC contraction/coronary spasm being features of ‘no-reflow’ (NRF) and microvascular obstruction (MVO). At present, there are limited therapies to improve ischemic MVO outcomes, and prognosis is poor. Hypothesis: Using a rat model, we hypothesised that, i) ROCK2 mRNA is expressed in myocardium and coronary vasculature and ii) The selective ROCK2 inhibitor KD025, would reduce infarct size (IS%) and NRF% (MVO) following I/R. Methods: RNA scope in-situ hybridisation was performed with a fluorescent, multiplex assay for ROCK1/2 & VSMC mRNA in myocardium and coronary vasculature. Male SD rats underwent in-vivo myocardial infarction with 30min ischemia, 180min reperfusion. 15min prior to reperfusion, the ROCK inhibitors Fasudil and KD025 or vehicle (DMSO) were administered i.p. For IS%, myocardium was stained with TTC, and regions not perfused with 1.5% Thioflavin S (NRF%), were visualised under UV light. Results: RNAscope confirmed the presence of ROCK2 mRNA within myocardium and VSMC of coronary arteries. Fasudil (10mg/kg) vs control significantly reduced regional IS% (30.3±4.4 vs52.9±3.8,p=0.02, n=15) and area of NRF% (12.4±2.8 vs28.6±2.2, p=0.001, n=15). However, there was significant hypotension;- Mean BP (mmHg) (72±3.9) vs control (84±2.3, p=0.007). KD025 (100mg/kg) did not reduce IS%, but significantly reduced the area of NRF% vs control (18.4±2.8 vs28.6±2.2,p=0.02, n=14) without hypotensive effect. Conclusions: Our results suggest that ROCK2 may be a prospective target in the management of coronary circulation reperfusion injury and ischemic MVO.

A Murine Calvarial Defect Model for the Investigation of the Osteogenic Potential of Fetal Umbilical Cord Stem Cells in Alveolar Cleft Repair

Journal of Oral and Maxillofacial Surgery

2022 Sep 01

Stanton, E;Sanchez, J;Kondra, K;Jimenez, C;Urata, M;Hammoudeh, J;
| DOI: 10.1016/j.joms.2022.07.012

Background: The standard graft material for alveolar cleft repair (ACR) is autogenous iliac crest. However, a promising alternative potential graft adjunct - newborn human umbilical cord mesenchymal stem cells (h-UCMSC) - has yet to be explored in vivo. Their capacity for selfrenewal, multipotent differentiation, and proliferation allows h-UCMSC to be harnessed for regenerative medicine. Our study seeks to evaluate the efficacy of using tissue-derived hUCMSC and their osteogenic capabilities in a murine model to improve ACR. Methods: Foxn1 mice were separated into three groups with the following calvarial defects: (1) no-treatment (empty defect; n=6), (2) poly (D,L-lactide-co-glycolide) (PLGA) scaffold (n=6), and (3) h-UCMSC with PLGA (n=4). Bilateral 2-mm diameter parietal bone critical-sized defects were created using a dental drill. Micro-CT imaging occurred at 1, 2, 3, and 4 weeks postoperatively. The mice were euthanized 4 weeks postoperatively for RNAscope analysis, immunohistochemistry, and histology. Results: No mice experienced complications during the follow-up period. Micro-CT and histology demonstrated that the no-treatment (1) and PLGA-only (2) defects remained patent without significant defect size differences across groups. In contrast, the h-UCMSC with PLGA group (3) had significantly greater bone fill on micro-CT and histology. Conclusions: We demonstrate a successful calvarial defect model for the investigation of hUCMSC-mediated osteogenesis and bone repair. Furthermore, evidence reveals that PLGA alone has neither short-term effects on bone formation nor any unwanted side effects, making it an 3 attractive scaffold. Further investigation using h-UCMSC with PLGA in larger animals is warranted to advance future translation to patients requiring ACR. Clinical Relevance Statement: Our results demonstrate a successful murine calvarial defect model for the investigation of h-UCMSC-mediated osteogenesis and bone repair and provide preliminary evidence for the safe and efficacious use of this graft adjunct in alveolar cleft repair.

P.188 Early growth and metabolic abnormalities in zebrafish and cellular models of SELENON-related myopathy

Neuromuscular Disorders

2022 Oct 01

Barraza-Flores, P;Moghadaszadeh, B;Mitchell, B;Troiano, E;Mansur, A;Gupta, V;
| DOI: 10.1016/j.nmd.2022.07.331

SELENON-Related Myopathy (SELENON-RM) is a rare genetic disease caused by recessive mutations of the SELENON gene. It is characterized by the development of rigid spine, axial muscle weakness, and respiratory insufficiency. The most common histopathological feature in SELENON-RM patients is the presence of minicores in skeletal muscle biopsies, which are concentrated areas of mitochondrial depletion within fibers. Natural history data suggest that insulin-resistance as well as altered body mass index (BMI) are correlated with SELENON-RM prognosis. There is no cure or effective treatment for SELENON-RM. The SELENON gene encodes selenoprotein-N, an endoplasmic reticulum (ER) membrane glycoprotein with reducing catalytic activity. Selenoprotein-N has been shown to activate SERCA channels by reducing it at low Ca2+ concentrations in the ER. Additionally, it has also been shown to colocalize with Mitochondrial Associated Membranes (MAM), which are vital for mitochondrial function. However, the molecular mechanism(s) by which selenoprotein-N deficiency causes SELENON-RM is still unclear. A particular challenge has been the lack of cellular or animal models that exhibit readily assayable phenotypes. In this project, we aim to identify cellular and animal models suited for high throughput drug screening while elucidating the molecular mechanism of the disease. To do so, we tested selenoprotein-N deficient cells and zebrafish selenon-null models. Using different measures of metabolism, we found that Selenon-KO C2C12 cells and primary myoblasts isolated from Selenon-KO mice were metabolically impaired. We also assessed several phenotyping outcomes in zebrafish models, from embryonic development to adulthood. Our results showed muscle weakness during early development as well as reduced growth during the larval stage. Together, these data establish the potential for selenoprotein-N deficient cells and zebrafish as models for the discovery of therapeutic targets for SELENON-RM.

P.134a Phase 1/2a trial of delandistrogene moxeparvovec in patients with DMD: 4-year update

Neuromuscular Disorders

2022 Oct 01

Mendell, J;Sahenk, Z;Lehman, K;Lowes, L;Reash, N;Iammarino, M;Alfano, L;Lewis, S;Church, K;Shell, R;Potter, R;Griffin, D;Pozsgai, E;Hogan, M;Hu, L;Mason, S;Darton, E;Rodino-Klapac, L;
| DOI: 10.1016/j.nmd.2022.07.251

Delandistrogene moxeparvovec (SRP-9001) is an investigational gene transfer therapy developed for targeted skeletal and cardiac muscle expression of micro-dystrophin (a shortened, functional dystrophin protein). The objective of this phase 1/2a, single-dose, open-label clinical trial (NCT03375164) is to evaluate the safety of systemic delivery of delandistrogene moxeparvovec in patients with Duchenne muscular dystrophy (DMD). Four ambulatory patients with DMD (≥4 to ≤8 years old) were enrolled. Patients were given an intravenous infusion of delandistrogene moxeparvovec at a dose of 2.0x1014 vg/kg (supercoiled qPCR, linear plasmid standard equivalent of 1.33x1014 vg/kg) and prednisone (1 mg/kg/day) 1 day pre- to 30 days post-gene delivery. The primary outcome measure is safety. The secondary outcome measures include micro-dystrophin expression in pre- and post-muscle biopsies (Week 12 post-infusion). Key efficacy outcome measures include North Star Ambulatory Assessment (NSAA) and timed function tests. Previously, data from 3 years post-treatment were presented. Treatment-related adverse events (TRAEs) were mild to moderate, occurred mostly in the first 90 days of treatment, and all resolved. No serious adverse events (AEs), study discontinuations, or AEs associated with clinically relevant complement activation were reported. All patients demonstrated a clinically meaningful improvement on NSAA. Patients treated with delandistrogene moxeparvovec generally maintained muscle strength (Time to Rise and 4-stair Climb) and showed improvement in ambulation ability (10-metre and 100-metre Walk/Run) from baseline to Year 3. The observed safety profile and the enduring response following treatment provide proof of concept for continuation of clinical trials assessing delandistrogene moxeparvovec using single-dose gene therapy in patients with DMD. We present the latest long-term (4-year) safety and functional data from this study.

The Alternative Matrisome: alternative splicing of ECM proteins in development, homeostasis and tumor progression

Matrix biology : journal of the International Society for Matrix Biology

2022 May 07

Rekad, Z;Izzi, V;Lamba, R;Ciais, D;Van Obberghen-Schilling, E;
PMID: 35537652 | DOI: 10.1016/j.matbio.2022.05.003

The extracellular matrix (ECM) is a fundamental component of the tissue of multicellular organisms that is comprised of an intricate network of multidomain proteins and associated factors, collectively known as the matrisome. The ECM creates a biophysical environment that regulates essential cellular processes such as adhesion, proliferation and migration and impacts cell fate decisions. The composition of the ECM varies across organs, developmental stages and diseases. Interestingly, most ECM genes generate transcripts that undergo extensive alternative splicing events, producing multiple protein variants from one gene thus enhancing ECM complexity and impacting matrix architecture. Extensive studies over the past several decades have linked ECM remodeling and expression of alternatively spliced ECM isoforms to cancer, and reprogramming of the alternative splicing patterns in cells has recently been proposed as a new hallmark of tumor progression. Indeed, tumor-associated alternative splicing occurs in both malignant and non-malignant cells of the tumor environment and growing evidence suggests that expression of specific ECM splicing variants could be a key step for stromal activation. In this review, we present a general overview of alternative splicing mechanisms, featuring examples of ECM components. The importance of ECM variant expression during essential physiological processes, such as tissue organization and embryonic development is discussed as well as the dysregulation of alternative splicing in cancer. The overall aim of this review is to address the complexity of the ECM by highlighting the importance of the yet-to-be-fully-characterized "alternative" matrisome in physiological and pathological states such as cancer.

RTOG-0129 risk groups are reproducible in a prospective multicenter heterogeneously treated cohort

Cancer

2021 Jun 18

Fakhry, C;Tewari, SR;Zhang, L;Windon, MJ;Bigelow, EO;Drake, VE;Rooper, LM;Troy, T;Ha, P;Miles, BA;Mydlarz, WK;Eisele, DW;D'Souza, G;
PMID: 34143891 | DOI: 10.1002/cncr.33682

Recursive partitioning analysis (RPA) from the Radiation Therapy Oncology Group (RTOG)-0129 has identified a low-risk group of patients with oropharynx cancer (OPC) who might benefit from therapeutic de-intensification. These risk groups have not yet been reproduced in an independent cohort treated heterogeneously. Therefore, the objective of this analysis was to validate the RPA risk groups and examine the prognostic impact of novel factors.Patients with OPC were enrolled in a prospective study at 3 academic medical centers from 2013 to 2018. Medical record abstraction was used to ascertain clinical variables including staging and survival according to the 7th edition of the American Joint Committee on Cancer (AJCC) Cancer Staging Manual. Human papillomavirus-positive tumor status was determined by p16 immunohistochemistry and/or HPV RNA in situ hybridization. Kaplan-Meier and log-rank methods were used to compare survival. Cox proportional hazards were used to generate univariate and multivariable hazard ratios (HRs).Median follow-up time was 3.2 years. The low-, intermediate-, and high-risk groups had significant differences in 2-year overall survival (OS, 99.1%; 95% CI, 94.4%-99.9% vs OS, 93.0%; 95% CI, 74.7%-98.2% vs OS, 80.0%; 95% CI, 40.9%-94.6%; Poverall = .0001) and 2-year progression-free survival (PFS, 97.5%; 95% CI, 92.4%-99.2% vs PFS, 89.3%; 95% CI, 70.3%-96.4% vs PFS, 80.0%; 95% CI, 40.9%-94.6%; Poverall < .002). After adjustment for age, sex, and level of educational attainment, OS and PFS were significantly lower for the intermediate- (OS adjusted hazard ratio [aHR], 5.0; 95% CI, 1.0-23.0; PFS aHR, 3.4; 95% CI, 1.0-11.5), and high- (OS aHR, 7.3; 95% CI, 1.4-39; PFS aHR, 5.0; 95% CI, 1.2-21.6) risk groups compared with the low-risk group. Lower education was also independently significantly associated with worse OS (aHR, 8.9; 95% CI, 1.8-44.3) and PFS (aHR, 3.1; 95% CI, 1.0-9.6).In patients with OPC, the RTOG-0129 RPA model is associated with OS and PFS in a heterogeneously treated cohort.

Psoriasis-like Inflammation Induced in an Air-pouch Mouse Model

In vivo (Athens, Greece)

2021 Mar 13

Charitidis, FT;Damlund, DSM;Koch, J;
PMID: 34182473 | DOI: 10.21873/invivo.12467

The pathway of initiation of psoriasis comprises the differentiation and infiltration of T-helper 17 (Th17) cells into the skin, characterized by the production of interleukin 17A and 17F (IL-17A/IL-17F) among other cytokines, resulting in a downstream cascade of events. Due to the lack of simplicity in psoriasis models, we aimed to develop an easily and rapidly inducible mouse model for the IL-23/IL-17 pathway with quick readouts from a straightforward lavaging process and with detectable cytokine levels.We utilized the 6-day air-pouch mouse model, injected with a combination of anti-CD3, IL-23 and IL-1β. At 24, 48 and 72 h, intra-pouch secretion of IL-17A, IL-17F and C-X-C motif chemokine ligand 1 were measured. Skin biopsies were collected and immune cell infiltration evaluated, and intra-pouch immune cells were isolated and analyzed.The combination of anti-CD3, IL-23 with and without IL-1β significantly increased intra-pouch levels of IL-17A/IL-17F at 24 and 72 h, triggering a downstream production of C-X-C motif chemokine ligand 1. The cytokines were detectable even 72 h post-induction. T-cell receptor beta was down-regulated on CD4+ and CD8+ T-cells, indicating intra-pouch T-cell activation. Αnti-CD3 induced CD3+ cell migration into the subcutis and the lining tissue surrounding the cavity of the air pouch, where in the latter, a similar distribution pattern of Il17a mRNA-expressing cells was also observed. However, no Th17 cell differentiation nor changes in IL-17A+ granulocytes were observed.The induced air-pouch mouse model induced with a cocktail of anti-CD3, IL-23 with or without IL-1β is able to mirror the IL-23/IL-17 axis of psoriasis-like inflammation characterized by immune cell infiltration and cytokine secretion.

Axin2+ peribiliary glands in the periampullary region generate biliary epithelial stem cells that give rise to ampullary carcinoma

Gastroenterology

2021 Jan 16

Hayata, Y;Nakagawa, H;Kurosaki, S;Kawamura, S;Matsushita, Y;Hayakawa, Y;Suzuki, N;Hata, M;Tsuboi, M;Kinoshita, H;Miyabayashi, K;Mizutani, H;Nakagomi, R;Ikenoue, T;Hirata, Y;Arita, J;Hasegawa, K;Tateishi, K;Koike, K;
PMID: 33465373 | DOI: 10.1053/j.gastro.2021.01.028

Peribiliary glands (PBGs), clusters of epithelial cells residing in the submucosal compartment of extrahepatic bile ducts, have been suggested as biliary epithelial stem/progenitor cell niche; however, evidence to support this claim is limited due to a lack of PBG-specific markers. We therefore sought to identify PBG-specific markers to investigate the potential role of PBGs as stem/progenitor cell niches, as well as an origin of cancer. We examined the expression pattern of the Wnt target gene Axin2 in extrahepatic bile ducts. We then applied lineage tracing to investigate whether Axin2-expressing cells from PBGs contribute to biliary regeneration and carcinogenesis using Axin2-CreERT mice. Wnt signaling activation, marked by Axin2, was limited to PBGs located in the periampullary region. Lineage tracing revealed that Axin2-expressing periampullary PBG cells are capable of self-renewal and supplying new biliary epithelial cells (BECs) to the luminal surface. Additionally, the expression pattern of Axin2 and the mature ductal cell marker CK19 was mutually exclusive in periampullary region, and fate tracing of CK19+ luminal surface BECs revealed gradual replacement by CK19- cells, further supporting the continuous replenishment of new BECs from PBGs to the luminal surface. We also found that Wnt signal enhancer R-spondin3 secreted from Myh11-expressing stromal cells, corresponding to human sphincter of Oddi, maintained the periampullary Wnt signal-activating niche. Notably, introduction of PTEN deletion into Axin2+ PBG cells, but not CK19+ luminal surface BECs, induced ampullary carcinoma whose development was suppressed by Wnt inhibitor. A specific cell population receiving Wnt-activating signal in periampullary PBGs functions as biliary epithelial stem/progenitor cells and also cellular origin of ampullary carcinoma.

Factors for risk stratification of patients with actinic keratosis using integrated analysis of clinicopathological features and gene expression patterns

The Australasian journal of dermatology

2023 Jan 16

Kim, HN;Kim, H;Gim, JA;Baek, YS;Kim, A;Kim, C;
PMID: 36645414 | DOI: 10.1111/ajd.13965

Actinic keratosis (AK) is considered as precursor lesion of invasive squamous cell carcinoma. Molecular studies on AK are limited because of too small size of the biopsy specimen to obtain enough DNA or RNA.Twenty biopsy cases of AK, followed by second same-sited biopsies, were included. Ten cases were diagnosed with total regression (regression group), while the other 10 were diagnosed with invasive carcinoma (progression group) in the follow-up biopsies. Using digital spatial profiling (DSP) technology, whole-gene expression analysis defined by specific regions of interest was performed for all 20 cases. After the clinicopathological features were assessed, separate and integrated analyses of these features and gene expression patterns were performed using machine-learning technology. All analyses were performed on both lesion keratinocytes (KT) and infiltrated stromal lymphocytes (LC).Among the 18,667 genes assessed, 33 and 72 differentially expressed genes (DEGs) between the regression and progression groups were found in KT and LC respectively. The primary genes distinguishing the two groups were KRT10 for KT and CARD18 for LC. Clinicopathological features were weaker in risk stratification of AK progression than the gene expression patterns. Pathways associated with various cancers were upregulated in the progression group of KT, whereas the nucleotide-binding oligomerization domain (NOD)-like receptor signalling pathway was upregulated in the progression of LC.Gene expression patterns were effective for risk stratification of AK progression, and their distinguishing power was higher than that of clinicopathological features.

Aortic Cellular Diversity and Quantitative Genome-Wide Association Study Trait Prioritization Through Single-Nuclear RNA Sequencing of the Aneurysmal Human Aorta

Arteriosclerosis, thrombosis, and vascular biology

2022 Nov 01

Chou, EL;Chaffin, M;Simonson, B;Pirruccello, JP;Akkad, AD;Nekoui, M;Lino Cardenas, CL;Bedi, KC;Nash, C;Juric, D;Stone, JR;Isselbacher, EM;Margulies, KB;Klattenhoff, C;Ellinor, PT;Lindsay, ME;
PMID: 36172868 | DOI: 10.1161/ATVBAHA.122.317953

Mural cells in ascending aortic aneurysms undergo phenotypic changes that promote extracellular matrix destruction and structural weakening. To explore this biology, we analyzed the transcriptional features of thoracic aortic tissue.Single-nuclear RNA sequencing was performed on 13 samples from human donors, 6 with thoracic aortic aneurysm, and 7 without aneurysm. Individual transcriptomes were then clustered based on transcriptional profiles. Clusters were used for between-disease differential gene expression analyses, subcluster analysis, and analyzed for intersection with genetic aortic trait data.We sequenced 71 689 nuclei from human thoracic aortas and identified 14 clusters, aligning with 11 cell types, predominantly vascular smooth muscle cells (VSMCs) consistent with aortic histology. With unbiased methodology, we found 7 vascular smooth muscle cell and 6 fibroblast subclusters. Differentially expressed genes analysis revealed a vascular smooth muscle cell group accounting for the majority of differential gene expression. Fibroblast populations in aneurysm exhibit distinct behavior with almost complete disappearance of quiescent fibroblasts. Differentially expressed genes were used to prioritize genes at aortic diameter and distensibility genome-wide association study loci highlighting the genes JUN, LTBP4 (latent transforming growth factor beta-binding protein 1), and IL34 (interleukin 34) in fibroblasts, ENTPD1, PDLIM5 (PDZ and LIM domain 5), ACTN4 (alpha-actinin-4), and GLRX in vascular smooth muscle cells, as well as LRP1 in macrophage populations.Using nuclear RNA sequencing, we describe the cellular diversity of healthy and aneurysmal human ascending aorta. Sporadic aortic aneurysm is characterized by differential gene expression within known cellular classes rather than by the appearance of novel cellular forms. Single-nuclear RNA sequencing of aortic tissue can be used to prioritize genes at aortic trait loci.

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	Description
sense Example: Hs-LAG3-sense	Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe.
Intron# Example: Mm-Htt-intron2	Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection
Pool/Pan Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G)	A mixture of multiple probe sets targeting multiple genes or transcripts
No-XSp Example: Hs-PDGFB-No-XMm	Does not cross detect with the species (Sp)
XSp Example: Rn-Pde9a-XMm	designed to cross detect with the species (Sp)
O# Example: Mm-Islr-O1	Alternative design targeting different regions of the same transcript or isoforms
CDS Example: Hs-SLC31A-CDS	Probe targets the protein-coding sequence only
EnEm	Probe targets exons n and m
En-Em	Probe targets region from exon n to exon m
Retired Nomenclature
tvn Example: Hs-LEPR-tv1	Designed to target transcript variant n
ORF Example: Hs-ACVRL1-ORF	Probe targets open reading frame
UTR Example: Hs-HTT-UTR-C3	Probe targets the untranslated region (non-protein-coding region) only
5UTR Example: Hs-GNRHR-5UTR	Probe targets the 5' untranslated region only
3UTR Example: Rn-Npy1r-3UTR	Probe targets the 3' untranslated region only
Pan Example: Pool	A mixture of multiple probe sets targeting multiple genes or transcripts

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