NAD+ Supplementation Strengthens the Thoracic Aorta by Modulating YAP/SOX9-dependent Mechanotransduction and Extracellular Matrix Remodeling

Investigator: Hao Yin

Supervisor: J. Geoffrey Pickering

Institution: Western University

Address: 1151 Richmond Street, London, Ontario, Canada, N6A 3K7

1. Overview

1.1. Scientific rationale and importance:

Thoracic aortic aneurysm (TAA) is characterized by medial degeneration with progressive loss of organized fibrillar collagen and pathologic accumulation of proteoglycan/glycosaminoglycans (GAGs), leading to biomechanical failure and catastrophic dissection or rupture, with over 50% mortality. One major pathogenic mechanism of TAA is the defective mechanosensing/mechanotransduction of medial smooth muscle cells (SMCs). Our group has demonstrated that human dilated aortas exhibit reduced NAMPT immunostaining, and SMC‑specific Nampt knockout (KO) mice show a >40 % reduction of medial NAD⁺ (nicotinamide adenine dinucleotide) and are highly susceptible to angiotensin II–induced medial hemorrhage and dissection¹. Moreover, a recent study showed that NAD⁺ biosynthetic enzymes are required for mitochondrial proline biosynthesis and collagen maturation². Sirtuin 1 (Sirt1) is an NAD⁺‑dependent deacetylase that exerts anti‑oxidant and anti‑inflammatory effects in the aortic wall; its activation protects against aortic aneurysm and dissection and reduces matrix metalloproteinase activity³. Given that NAD⁺ supplementation is on active trials targeting vascular aging, heart failure, and neurodegenerative diseases, understanding its vascular mechanisms is important. However, the transcriptional mechanisms by which NAD⁺ shapes aortic ECM gene expression—particularly under mechanical and/or oxidative stress—remain undefined.

Mechanistically, we propose that enhancing NAD⁺ production promotes a collagen‑rich, GAG-low, mechanically stable ECM and reinforces mechanotransduction in aortic SMCs by activating the Sirt1–YAP cascade and repressing SOX9 expression. Sirt1 deacetylates YAP, promoting its nuclear localization and activating transcription of fibrillar collagen genes. When NAD⁺ levels fall, Sirt1 activity decreases; YAP becomes inactivated, leading to SOX9‑dependent expression of proteoglycan/GAGs and downregulation of fibrillar collagens. Supportive evidence for this model includes (1) our previous publication and pilot data showing that SMC-specific Nampt KO mice receiving angiotensin II are susceptible to ascending aortic hemorrhage, dissection and rupture, correlated with dampened collagen fibers and excess GAG deposits; and (2) literature on the roles of YAP and its homolog TAZ in collagen biosynthesis⁴, and SOX9 in proteoglycan/GAG biosynthesis in SMCs or other mesenchymal lineages⁵. This proposal will employ 3D human SMC organoids for in vitro assays, which recapitulate cell-cell contacts, matrix deposition, and mechanical features that are either absent or exaggerated in 2D cell culture. Successful completion of this project will uncover a novel metabolo-mechanical control of ECM transcriptional programs in the aorta. Note that this project is part of an ongoing multi-year project in the Pickering lab. Therefore, this proposal does NOT aim to generate comprehensive experimental evidence to cover all necessary and sufficient details. Instead, the anticipated findings from each of the 3 Aims will be rigorous, stand-alone evidence that serve as the foundation for more integrated and in-depth research of the Pickering lab (see Expanded Future Plan for details), which potentially identify NAD⁺ precursor administration as a promising therapeutic target to promote the regeneration of vasculature and musculature.

1.2. Facilities/ethics approvals:

Required infrastructure and core services for RNA sequencing and confocal imaging are available at Western University for the proposed 6-month project. Ethics approvals for the use of human tissue and cells are ready in place.

1.3. Estimated timeline: 6 months in total

Month 1-3: Aim 1 – NAD⁺ bioavailability and collagen-GAG switch in human SMC spheroids;

Month 4-5: Aim 2 – NAD+ bioavailability and mechanotransduction in human SMC spheroids;

Month 6: Aim 3 – Immunohistochemical validation of key signaling mediators in human aortas.

1.4. Data sharing commitment:

All analysis R/Python codes and raw/processed sequencing datasets will be shared in a public repository (Github, GEO or SRA).

2. Introduction

2.1. Background

Thoracic aortic aneurysm and dissection (TAAD) is a life‑threatening condition characterized by aortic dilation, defective mechanosensing of medial SMCs, and mechanical weakening of the extracellular matrix (ECM). Healthy SMCs adhere to ECM and sense the stiffness and tension of the aortic wall; failing in mechanosensing not only dampens the force transmission, but also causes conversion of SMCs into a matrix-degradative phenotype, contributing to the degradation of elastic lamina, disorganization of collagen fibers, and accumulation of proteoglycan/GAGs⁶. GAGs at normal levels maintain aortic tissue hydration and resistance to compressive forces. However, ectopic pools of GAGs decrease tensile strength and increase local stress and intralamellar swelling pressure, which may initiate delaminations and propagate dissections⁷. On the other hand, fibrillar collagen (type I and III collagens) is crucial to the structure and biomechanics of the aortic wall; it acts as the major load-bearing matrix that provides tensile strength and prevents rupture of the injured aorta^8,9.

Importantly, both mechanosensing and collagen anabolism require NAD⁺. Genetic or pharmacological perturbation of NAD+ biosynthesis (targeting NAMPT or NRK2) compromises adhesion-based mechanosensing in myocytes. For example, our group reported that NAMPT promotes directional persistence of SMC migration via local activation of Cdc42¹⁰. NAD⁺ depletion in mitochondria, induced by NAD⁺ transporter SLC25A51 deficiency, limits proline biosynthesis in mitochondria and subsequent collagen III turnover². Concordantly, thoracic aortas in mice with SMC-specific Nampt KO are refractory to angiotensin II-induced collagen fibrillogenesis (see Pilot data for details), and prone to dissection¹. Moreover, NAMPT protein expression was inversely related to ascending aortic diameter in patients with TAA¹. Taken together, these studies support a critical role of NAD⁺ bioavailability in maintaining SMC mechanosensing/mechanotransduction and stress-adaptive ECM remodeling. Beyond mitochondrial metabolism, NAD⁺ is also a nuclear signaling message. The NAD⁺ salvage enzymes, NAMPT or NMNAT1, locally sustain Sirtuin 1 (Sirt1) activity at target gene promoters, directly coupling NAD⁺ bioavailability to transcriptional outputs. In parallel, PARP1 utilizes NAD⁺ to ADP-ribosylate chromatin-associated proteins. These important lines of evidence strongly suggest a second route by which NAD⁺ fluctuations could directly reprogram ECM gene transcription.

Important candidate NAD⁺-related transcriptional factors include YAP (Yes Associated Protein 1) and TAZ (Transcriptional Coactivator with PDZ-Binding Motif), which are central mechanotransduction regulators downstream of Sirtuin 1 (Sirt1), an NAD⁺‑dependent deacetylase. Sirt1-YAP/TAZ axis plays an essential role in fluid shear sensing in epithelial or endothelial cells^11,12 and stiffness sensing in pulmonary arterial SMCs¹³. YAP/TAZ controls collagen gene expression in SMCs and mesenchymal lineage cells⁴. Reciprocally, collagen fibrillation-induced vascular stiffening mechanoactivates YAP/TAZ¹⁴, creating a feedforward cycle that exacerbates peri-arterial fibrosis in the lung. In SMCs, YAP/TAZ act as mechanoprotective factors: SMC‑specific ablation of YAP/TAZ in adult mice leads to loss of contractile phenotype of vascular and visceral SMCs, correlated with rapid aneurysm formation⁵ and colonic pseudo-obstruction¹⁵. However, it remains unclear whether attenuated collagen fibers account for these smooth muscle phenotypes.

Intriguingly, the onset of aortic aneurysm upon induced YAP/TAZ deficiency correlates with early accumulation of GAGs, attributed to increased SOX9 and its target gene Acan/aggrecan⁵, a major proteoglycan core protein in TAA¹⁶. In addition, SOX9 translates stiffness and contractile tension into ECM-related transcriptional outputs in SMCs and tenocytes, respectively^17,18. These findings highlight the potential dual roles of YAP/TAZ and SOX9 in SMC mechanotransduction and collagen-GAG matrix switch. Yet, the regulatory relationship between NAD+–SIRT1 signaling, YAP/TAZ- and SOX9-driven ECM programs remains unexplored.

Our central hypothesis is that NAD⁺ supplementation, via supplying nicotinamide riboside (NR), enhances Sirt1 activity in aortic SMCs, promoting YAP/TAZ- and SOX9‑dependent mechanotransduction and collagen-GAG matrix program

2.2. Specific hypotheses

H1: NAD⁺ supplementation in human aortic SMC spheroids will activate SIRT1, which works upstream of the YAP/TAZ-SOX9 control of the ECM transcriptional program.
H2: NAD⁺ supplementation coordinates with contractile force transmission machinery to drive collagen-rich, GAG-low ECM in SMC spheroids.
H3: Human aneurysmal ascending aortas exhibit reduced NAMPT and YAP/TAZ immunoreactivity with increased SOX9 and GAG accumulation.

2.3. Impact statement

Understanding how NAD⁺ modulates aortic ECM dynamics and the upstream transcriptional regulatory factors could identify a novel mechanism linking metabolic health with aortic wall mechanical functions and structural integrity. If NAD⁺ supplementation enhances collagen-rich ECM and suppresses GAG accumulation, NAD⁺ precursors (e.g., nicotinamide riboside/NR), which are in active phase I/II trials, may represent a low‑risk therapeutic avenue to stabilize dilated thoracic aortas. Conversely, if NAD⁺ supplementation inadvertently increases GAG deposition or compromises SMC contractile phenotype and/or mechanotransduction, these findings would caution against indiscriminate use of NAD⁺ boosters in patients with at-risk aortas. Even though no significant impact was observed, our data might still serve as a line of evidence for the macrovascular safety of NAD⁺ supplementation.

3. Methods

This project is organized into 3 specific aims to be completed over a 6-month period, as an integral part of an ongoing large project in the Pickering lab. A brief statement on how the current data empowers an expanded, future research plan is included at the end of this section.6 months

Aim 1: To determine how NAD⁺ supplementation modulates the activities of SIRT1, YAP/TAZ, and SOX9, and the downstream ECM transcriptional programs - fibrillar collagen versus proteoglycan/GAG genes.

Sample: Primary human ascending aortic SMCs (healthy donors and patients with TAA) at passage 3 to 6 will be used to generate SMC organoids/spheroids using 25,000 cells per well using a cell-repellent, U-bottom 96-well plate, n=6 for each subject group. Only sporadic TAA patients will be included in this proposal. We will balance the sex when possible, as sporadic TAA patients have an approximate 4:1 male-to-female ratio. Our preliminary study showed that spheroids form within 24 hours and are detectable with GAG deposits (using chondroitin sulfate immunofluorescence) at around 72 hours.

· Nicotinamide riboside (NR) regimen and NAD⁺ bioavailability: NR across a range of concentrations (0.1, 1, 10, 100, 1000 μM) will be added at 24 hours of spheroid formation. This concentration range has been tested on 2D SMC culture in the Pickering lab. By day 3, spheroids will be harvested to (i) quantify NAD⁺/NADH ratio and total NAD(H) level using a bioluminescent kit (Promega), (ii) evaluate cell viability using LIVE/DEAD Viability/Cytotoxicity Kit (calcein-AM + ethidium homodimer-1) (ThermoFisher Scientific), and (iii) measure spheroid size and shape.

· ECM program readouts: ECM transcripts will be assessed with quantitative PCR (qPCR). The collagen program covers genes encoding fibrillar collagens (COL1A1, COL1A2, COL3A1) and collagen-crosslinking enzymes (LOX, LOXL1-4, PLOD1-3, TGM2); the GAG program covers genes encoding proteoglycan core proteins (ACAN, VCAN, BGN), GAG biosynthetic enzymes (XYLT1, XYLT2, CSGALNACT1, CSGALNACT2, CHSY1, CHSY3, CHPF, CHPF2), and hyaluronan (non-sulfated GAG scaffold) synthases (HAS1, HAS2, HAS3). Spheroidal ECM protein deposition will be assessed with picrosirius red staining for collagen fiber and confocal-based immunofluorescent analysis for chondroitin sulfate (as a surrogate for GAG, see Pilot data for details). Note that picrosirius red-stained fibrils are fluorogenic and imageable with confocal microscopy¹⁹. Additionally, the contents of fibrillar collagen and GAG in spheroid homogenates will be quantified using the colorimetric Hydroxyproline Assay Kit (Sigma #MAK463) and the Blyscan sulfated GAG Assay Kit (Biocolor #B1000).

· The role of Sirt1: Sirt1 activity will be assessed using FLUOR DE LYS SIRT1 fluorometric assay kit (ENZO), and using immunofluorescent analysis for Sirt1 total abundance, subcellular localization (nuclei versus cytoplasm), and acetylation of canonical Sirt1 targets (p53 and FOXO1) and YAP¹². Inactivation of Sirt1 will be achieved using a pharmacological inhibitor (EX-527) or siRNA. Activation of Sirt1 will be achieved using a pharmacological activator (SRT-2104) or adenoviral overexpression of Sirt1.

· The roles of YAP/TAZ and SOX9: YAP/TAZ activity will be surrogated via immunofluorescent analysis for total abundance, inhibitory phospho-species (pYAP Ser127, pTAZ Ser89), and subcellular localization (nuclei versus cytoplasm). SOX9 activity will be surrogated via immunofluorescent analysis for total abundance and subcellular localization (nuclei versus cytoplasm). Inactivation of YAP/TAZ will be achieved using a pharmacological inhibitor (Verteporfin) or siRNAs. Activation of YAP/TAZ will be achieved using PY-60²⁰ or lentiviral overexpression of YAP-5SA or TAZ-4SA. ). Inactivation of SOX9 will be achieved using siRNA. Activation of SOX9 will be achieved using lentiviral overexpression of SOX9.

· Relative contributions of nuclear NAD⁺–SIRT1–YAP/TAZ versus mitochondrial NAD⁺–proline supply to fibrillar collagen assembly will be studied via (i) SLC25A51 siRNA in NR-treated spheroids; or (ii) exogenous proline supply in Sirt1- or YAP/TAZ-inhibited spheroids.

· Bulk RNA-seq will be conducted on spheroids treated with one effective concentration of NR or vehicle control with n=4 biological replicates. The effective NR concentration will be determined based on the statistically discernible alteration of collagen and/or GAG content.

Sample size and Power analysis: Based on our pilot data that NAMPT inhibition leads to increased spheroidal chondroitin sulfate signals, n=6 biological replicates (at patient/subject level) with 12 technical replicates (at individual spheroid level) can provide sufficient power to detect 30% difference in GAG content with α = 0.05, power = 0.90.

Planned Analyses:

Spheroid immunostaining and qPCR data will be analyzed using a linear mixed model. Fixed factors are subject group (healthy versus TAA) and intervention conditions (e.g., NR versus vehicle, Sirt1 siRNA versus negative control siRNA). Random factor is the individual subject. Multiplicity will be adjusted using Sidak’s post hoc test.

RNA-seq data will be analyzed in DESeq2. Genes with <10 counts in > 4 samples will be filtered out. The counts will then be modeled using DESeq2’s negative binomial generalized linear model with size factor normalization and dispersion estimation. A matched pair design (design = ~ subject + condition) will be used to test for differential expression via Wald tests. Multiple testing will be adjusted using the Benjamini-Hochberg procedure. Pathway enrichment analysis will be conducted using pre-ranked GSEA (gene set enrichment analysis). Upstream transcriptional activity will be predicted using ChEA-KG²¹.

Anticipated outcomes: Based on the pilot data and previous publications of our lab, we expect that spheroids made of TAA SMCs will exhibit lower NAD⁺ content, lower YAP/TAZ activity and fibrillar collagen content, and higher SOX9 activity and GAG content. Furthermore, we expect that NR supply could partially normalize these transcriptional and ECM defects of TAA spheroids. If Sirt1 inhibition does not block NR-induced collagen accumulation or GAG reduction, we will examine the involvement of other NAD⁺-dependent enzymes, prioritizing PARP1 or SIRT6, which possess YAP/TAZ-modifying capacities^22,23.

Aim 2: To determine how NAD⁺ supplementation coordinates with contractile force transmission machinery to drive GAG-low ECM in SMC spheroids.

Sample: Human SMC spheroids will be generated as described in Aim 1.

In this aim, we specifically hypothesize that NR supply improves contractile force transmission machinery, thereby repressing the SOX9-GAG axis.

· Contractile force transmission machinery will be assessed in healthy and TAA SMC spheroids treated with NR or vehicle control. Contractile phenotype will be assessed via immunofluorescent analysis for α-SMA, calponin, SMMHC, myocardin (master transcriptional co-activator of SMC contractile genes), serum response factor/SRF, total MLC2, and phospho Ser19 MLC2. Focal adhesion complex will be assessed via integrin β1, vinculin, total and phospho Tyr118 paxillin, and total and phospho Tyr397 FAK. Filamentous actin will be assessed using fluorophore-conjugated phalloidin.

· Cell-intrinsic stiffness will be attenuated using ROCK inhibitor (Y-27632) or non-muscle myosin II inhibitor (blebbistatin). Spheroid-level stiffness will be attenuated using LOX-collagen crosslink inhibitor (BAPN) or secondarily augmented using 500 kD dextran-based osmotic compression (with the same mass concentration of 50 kD dextran as control)²⁴. Considering the limited duration and scope of this proposal, we will not directly measure cellular or spheroidal stiffness using atomic force microscopy, which will be part of the expanded future research plan.

· YAP/TAZ expression/activity, SOX9 expression/activity, and proteoglycan/GAG transcripts and matrix products in SMC spheroids will be assessed as described in Aim 1.

Sample size and Power analysis: We assume the same sample size and power estimation as Aim 1.

Planned Analyses:

Spheroid immunostaining and qPCR data will be analyzed using a linear mixed model. Fixed factors are subject group (healthy versus TAA) and intervention conditions (e.g., NR versus vehicle, 500 kD versus 50 kD dextran). Random factor is the individual subject. Multiplicity will be adjusted using Sidak’s post hoc test.

Anticipated outcomes: We expect that spheroids made of TAA SMCs will exhibit a lower contractile phenotype, which can be partially rescued by NR. Furthermore, we expect that the beneficial effects of NR on contractile phenotype and SOX9-GAG cascade in TAA spheroids. If NR reduces GAG deposition despite actinomyosin inhibition, epistasis experiments targeting YAP/TAZ or SOX9 will delineate whether NR acts upstream or downstream of mechanical force transmission.

Aim 3: To validate key signaling mediators (YAP/TAZ, SOX9) in non-dilated and dilated human aortas.

Sample: Non-dilated (< 4 cm diameter) ascending aortas from healthy subjects and dilated (> 4.5 cm diameter) ascending aortas from subjects with sporadic TAA, n=10 per group.

· NAMPT (ThermoFisher Scientific # A700-058, rabbit monoclonal), YAP (Cell Signaling #14074, rabbit monoclonal), phospho Ser127 YAP (Cell Signaling #13008, rabbit monoclonal), TAZ (Cell Signaling #72804, rabbit monoclonal), phospho Ser89 TAZ (Creative Biolabs #CBMAB-CP2702-LY, rabbit monoclonal), or SOX9 (Cell Signaling #82630, rabbit monoclonal) will be assessed via immunofluorescent staining. Aortic GAG content will be detected via immunofluorescent staining for aggrecan (Sigma #MAB5284, mouse monoclonal), chondroitin sulfate (Sigma #SAB4200696, mouse monoclonal), or biotinylated hyaluronan binding protein (AMSbio #AMS.HKD-BC41). SMC and nuclei will be immunolabeled using SM α-actin antibody (Novus Biologicals # NB300-978, goat polyclonal) and DAPI, respectively. The combinations of primary antibodies of different hosts enable quadruple immunolabeling of NAMPT/YAP/TAZ/SOX9 together with GAG, SMC cytoplasm, and nuclei. The full-thickness aortic medial wall, where applicable, will be imaged under a confocal microscope.

Sample size and Power analysis: Based on our pilot data and previous publication¹ on NAMPT and YAP immunostaining on aortic sections, n=10 human aorta specimens can provide sufficient power to detect 30% difference in target proteins with α = 0.05, power = 0.80.

Planned Analyses:

The fluorescent density of signaling mediators (NAMPT/YAP/TAZ/SOX9) or GAG components (aggrecan/chondroitin sulfate/hyaluronan) in individual aortas will be averaged and compared between healthy and TAA groups using Welch’s t test or Mann-Whitney U test, depending on the data normality. For YAP/TAZ/SOX9, their nuclear density will also be computed at per-SMC base and averaged for individual aorta for between-group comparisons. To quantify the spatial coupling between each signaling mediator and each GAG component, the aortic media sampled will be binned into 20 μm x 20 μm metapixels, with integrated intensity computed per metapixel. Spatial association will be estimated in two approaches: (i) within-specimen Spearman’s correlations computed across metapixels, followed by comparing correlation strength (ρ) between groups; (ii) a mixed effects model at the level of metapixel, with a random intercept for subject. Furthermore, to adjust for the potential effects of age and sex on aortic ECM, a multivariable mixed effects model will be used to compute the associations of each GAG readout with each signaling mediator, with age and sex as covariates, and with a random intercept for subject. Multiplicity will be adjusted using the Benjamini-Hochberg procedure.

Anticipated outcomes: Based on our pilot data and previous publication¹, we expect that dilated human ascending aortas exhibit decreased immunoreactivities of NAMPT, YAP, TAZ, and increased immunoreactivities of phospho-YAP, phospho-TAZ, and SOX9, as compared with non-dilated aortas. Negative correlations are expected between NAMPT/YAP/TAZ with GAG components; Positive correlations are expected between phospho-YAP/phospho-TAZ/SOX9 and GAG components. These correlations are expected to be persistent after adjustment for age and sex.

Expanded, future research plan: This proposed work is expected to yield data that is to be incorporated into the multi-year ongoing project on NAD⁺ metabolism in TAAD. Several aspects of this proposal will be strengthened or supplemented by more collaborative experiments if positive data are obtained. (1) Spheroid mechanics, including stiffness and tensile/rupture strength, will be measured using atomic force microscopy in collaboration with CAMiLOD imaging facility at the University of Toronto. (2) More sporadic human TAA specimens will be studied, allowing for accounting for other risk factors (hypertension, smoking, and aortic diameter). In addition, other aortic etiologies, including bicuspid aortic valve-associated aortopathy and monogenic syndromes (Marfan or Loeys-Dietz syndrome), will be studied to evaluate the generalizability of NR benefits. (3) The in vivo benefits of NR will be validated in a mouse model of thoracic aortopathy, part of which can be found in a Pickering lab trainee’s thesis, as linked here - https://ir.lib.uwo.ca/etd/4788/ (4) Moreover, SMC signaling-metabolism coupling in thoracic aortas of NR-fed mice will be interrogated using spatial multiomics - spatial transcriptomics (Molecular Pathology Core Facility at Western University) and spatial metabolomics (CASBR at University of Florida).

Ethics and data management: Ethics approvals for the use of human tissues and cells are ready in place. All analysis R/Python codes and raw/processed sequencing datasets will be shared in a public repository (Github, GEO, or SRA).

4. Pilot Data (Optional)

Our hypothesis is strongly supported by pilot data from our group, as shown in the preliminary figure and data publicly available in the Pickering lab trainees’ thesis work.

(A, B) Immunohistochemical staining of NAMPT (brown) on sections of a non-dilated (left, A) and dilated (right, B) human ascending aorta. Zoom-in images at the bottom show dilated aorta exhibits reduced NAMPT immunoreactivities in both nuclear (arrows) and cytoplasmic (arrowhead) compartments of medial smooth muscle cells (SMCs). (C) Confocal micrographs of YAP (green) on sections of a non-dilated (left) and dilated (right) human ascending aorta. Medial SMCs were labeled with SM α-actin (green), elastin fibers with Alexa Fluor 633-conjugated hydrazide (magenta), and nuclei with DAPI (blue). Dilated aorta exhibits reduced YAP immunofluorescent signals in both nuclei and cytoplasm of medial SMCs. (D) Hematoxylin and eosin (HE)-stained sections of ascending aortas of angiotensin II-infused control (left) and SMC Nampt KO (right) mice. Intramedial hemorrhage can be found in dissected Nampt-null aortas. (E) HE (left, middle) and Movat’s pentachrome (right)-stained sections of ascending aortas of angiotensin II-infused control (left) and SMC Nampt KO (middle, right) mice. The white arrow indicates large focal GAG deposits in Nampt-null aortas. (F) Picrosirius red-stained sections of ascending aortas of saline- (left) or angiotensin II (middle, right)-infused control (left, middle) and SMC Nampt KO (right) mice. Collagen fibers were visualized using circularly polarized light microscopy. Both peri-elastin (white arrowheads) and between-lamellar (white arrows) collagen fibers are weakened in Nampt-null aortas. (G) Microarray analysis of isolated aortic media from angiotensin II-infused control and SMC Nampt KO mice. Heatmap depicts downregulated transcripts of fibrillar collagens and crosslinking enzyme (Col1a1, Col1a2, Col3a1, Lox) and upregulated transcripts of proteoglycan (Has2, Vcan) in Nampt-null aortas. (H) Confocal micrographs of human SMC spheroids treated with vehicle control (DMSO) or 10 nM FK866, NAMPT inhibitor, for 5 days. GAG was visualized via immunostaining for Chondroitin sulfate (red), the major GAG in aorta media. SMCs were labeled with SM α-actin (green), and nuclei with DAPI (blue). Inhibition of NAMPT activity led to an increase in intercellular GAG accumulation. Quantitative data can be found in the thesis of Dr. Alanna Watson, as linked - https://ir.lib.uwo.ca/etd/5060/

5. Budget

Note: All personnel costs are covered by active funds. Primary human SMCs, all PCR reagents and primers, most antibodies, and all histological/immunofluorescent reagents are available, as this proposal constitutes part of an ongoing project in the Pickering lab.

6. References

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NAD+ Supplementation Strengthens the Thoracic Aorta by Modulating YAP/SOX9-dependent Mechanotransduction and Extracellular Matrix Remodeling

NAD+ Supplementation Strengthens the Thoracic Aorta by Modulating YAP/SOX9-dependent Mechanotransduction and Extracellular Matrix Remodeling

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