Lineage Tracing Mouse Models Research Articles

MLKL-mediated endothelial necroptosis drives vascular damage and mortality in systemic inflammatory response syndrome.

The hypersecretion of cytokines triggers life-threatening systemic inflammatory response syndrome (SIRS), leading to multiple organ dysfunction syndrome (MODS) and mortality. Although both coagulopathy and necroptosis have been identified as important factors in the pathogenesis of SIRS, the specific cell types that undergo necroptosis and the interrelationships between coagulopathy and necroptosis remain unclear. In this study, we utilized visualization analysis via intravital microscopy to demonstrate that both anticoagulant heparin and nonanticoagulant heparin (NAH) pretreatment protect mice against TNF-α-induced mortality in SIRS. Moreover, the deletion of Mlkl or Ripk3 resulted in decreased coagulation and reduced mortality in TNF-α-induced SIRS. These findings suggest that necroptosis plays a key role upstream of coagulation in SIRS-related mortality. Furthermore, using a genetic lineage tracing mouse model (Tie2-Cre;Rosa26-tdT), we tracked endothelial cells (ECs) and verified that EC necroptosis is responsible for the vascular damage observed in TNF-α-treated mice. Importantly, Mlkl deletion in vascular ECs in mice had a similar protective effect against lethal SIRS by blocking EC necroptosis to protect the integrity of the endothelium. Collectively, our findings demonstrated that RIPK3-MLKL-dependent necroptosis disrupted vascular integrity, resulting in coagulopathy and multiorgan failure, eventually leading to mortality in SIRS patients. These results highlight the importance of targeting vascular EC necroptosis for the development of effective treatments for SIRS patients.

BST2 induces vascular smooth muscle cell plasticity and phenotype switching during cancer progression.

Smooth muscle cell (SMC) plasticity and phenotypic switching play prominent roles in the pathogenesis of multiple diseases, but their role in tumorigenesis is unknown. We investigated whether and how SMC diversity and plasticity plays a role in tumor angiogenesis and the tumor microenvironment. We use SMC-specific lineage-tracing mouse models and single cell RNA sequencing to observe the phenotypic diversity of SMCs participating in tumor vascularization. We find that a significant proportion of SMCs adopt a phenotype traditionally associated with macrophage-like cells. These cells are transcriptionally similar to 'resolution phase' M2b macrophages, which have been described to have a role in inflammation resolution. Computationally predicted by the ligand-receptor algorithm CellChat, signaling from BST2 on the surface of tumor cells to PIRA2 on SMCs promote this phenotypic transition; in vitro SMC assays demonstrate upregulation of macrophage transcriptional programs, and increased proliferation, migration, and phagocytic ability when exposed to BST2. Knockdown of BST2 in the tumor significantly decreases the transition towards a macrophage-like phenotype, and cells that do transition have a comparatively higher inflammatory signal typically associated with anti-tumor effect. As BST2 is known to be a poor prognostic marker in multiple cancers where it is associated with an M2 macrophage-skewed TME, these studies suggest that phenotypically switched SMCs may have a previously unidentified role in this immunosuppressive milieu. Further translational work is needed to understand how this phenotypic switch could influence the response to anti-cancer agents and if targeted inhibition of SMC plasticity would be therapeutically beneficial.

Brain-Engrafted Monocyte-derived Macrophages from Blood and Skull-Bone Marrow Exhibit Distinct Identities from Microglia.

Microglia are thought to originate exclusively from primitive macrophage progenitors in the yolk sac (YS) and to persist throughout life without much contribution from definitive hematopoiesis. Here, using lineage tracing, pharmacological manipulation, and RNA-sequencing, we elucidated the presence and characteristics of monocyte-derived macrophages (MDMs) in the brain parenchyma at baseline and during microglia repopulation, and defined the core transcriptional signatures of brain-engrafted MDMs. Lineage tracing mouse models revealed that MDMs transiently express CD206 during brain engraftment as CD206 + microglia precursors in the YS. We found that brain-engrafted MDMs exhibit transcriptional and epigenetic characteristics akin to meningeal macrophages, likely due to environmental imprinting within the meningeal space. Utilizing parabiosis and skull transplantation, we demonstrated that monocytes from both peripheral blood and skull bone marrow can repopulate microglia-depleted brains. Our results reveal the heterogeneous origins and cellular dynamics of brain parenchymal macrophages at baseline and in models of microglia depletion.

Combined lineage tracing and scRNA-seq reveal the activation of Sox9+ cells in renal regeneration with PGE2 treatment.

Uncovering mechanisms of endogenous regeneration and repair through resident stem cell activation will allow us to develop specific therapies for injuries and diseases by targeting resident stem cell lineages. Sox9+ stem cells have been reported to play an essential role in acute kidney injury (AKI). However, a complete view of the Sox9+ lineage was not well investigated to accurately elucidate the functional end state and the choice of cell fate during tissue repair after AKI. To identify the mechanisms of fate determination of Sox9+ stem cells, we set up an AKI model with prostaglandin E2 (PGE2) treatment in a Sox9 lineage tracing mouse model. Single-cell RNA sequencing (scRNA-seq) was performed to analyse the transcriptomic profile of the Sox9+ lineage. Our results revealed that PGE2 could activate renal Sox9+ cells and promote the differentiation of Sox9+ cells into renal proximal tubular epithelial cells and inhibit the development of fibrosis. Furthermore, single-cell transcriptome analysis demonstrated that PGE2 could regulate the restoration of lipid metabolism homeostasis in proximal tubular epithelial cells by participating in communication with different cell types. Our results highlight the prospects for the activation of endogenous renal Sox9+ stem cells with PGE2 for the regenerative therapy of AKI.

Abstract PO5-28-01: Dynamics of Adipocyte Progenitors in the Mammary Gland during Obesity

Abstract Being overweight or obese is associated with a higher risk of breast cancer (BC) and worse disease-free and overall survival compared to normal-weight BC patients. The underlying biological basis for this association is attributed to alterations in the endocrine and inflammatory milieu of adipose tissue. However, existing chemotherapy and endocrine therapy are not effective in obese BC patients. There is a critical need to pin down adipose tissue cell types that may govern increased BC risk and adverse outcome in obesity. Adipocyte progenitors are immature mesenchymal cells in adipose tissue that have adipocyte differentiation potential and reported to be dysfunctional in obesity. In previous research, we first reported mammary adipocyte progenitors (MAPs) expressing Platelet-Derived Growth Factor Receptor alpha (PDGFRα) in the stromal microenvironment that transition into epithelial lineages during expansion of the murine mammary epithelium. The contribution of MAPs to shaping a pro-tumorigenic mammary tissue milieu in obesity is not known. Here, using diet-induced obesity MAP reporter and lineage tracing mouse models combined with single-cell RNA sequencing, we reveal elevated PDGFRα+ MAPs in the local mammary stroma in obesity. MAPs in the obese mammary gland manifest alterations in proliferation, cell state and molecular mechanisms, generating an inflammatory, cancer-prone tissue microenvironment. Our findings uncover the MAP lineage as a key contributor to forging an aberrant mammary gland in obesity, providing insight into the potential utility of targeting this cell population for eradicating BC risk related to augmented adiposity. Citation Format: Sharon Kwende, Prashant Nuthalapati, Dun Ning, Jacqulene Sunder Singh, Purna Joshi. Dynamics of Adipocyte Progenitors in the Mammary Gland during Obesity [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-28-01.

Abstract 1050: Discrete Population Of Rare Smc Progenitor Gives Rise Exclusively To The Fibrous Cap

The fibrous cap of atherosclerotic plaques is essential for the plaque stability. Rupture of the fibrous cap leads to heart attacks and strokes, and causes tens of millions of deaths globally every year. Identifying and understanding the cellular origins and plasticity of the fibrous cap is critical to developing therapeutic strategies to stabilize the atherosclerotic plaques. Utilizing the lineage tracing mouse model, we demonstrated that fibrous cap cells arise from a predefined population of cells that expresses Notch3 at baseline. After pulse-labeling the Notch3 CreERT2 ; ROSA lsltdTomato ; Apoe -/- mice with tamoxifen before high fat diet and then feeding them with high fat diet for 16 weeks, Notch3 lineage traced cells stain positive for SMC markers (TAGLN, CNN1) and are nearly exclusively found at the fibrous cap in multiple atherosclerotic prone beds. Furthermore, Notch3-lineage traced SMCs and chondrogenic SMCs are mutually exclusive in the plaque, as demonstrated by the minimal overlap of tdTomato with chondrogenic SMC markers, including Col2a1 and Sox9. Not all fibrous caps are marked, which could reflect poor Cre efficiency or new cap cells that form later. Increase tamoxifen treatment frequencing throughout high fat diet (every 10 days) significantly improved labeling efficiency and led to fully labeled lesion cap of the whole aortic root plaque. Consistently, Notch3 + cells are committed to fibrous cap formation and excluded from the calcified portions of the lesion and acellular core. Collectively, these lineage tracing studies highlight previously unrecognized medial SMC heterogeneity in healthy vessels. Unique Notch3 + populations of SMCs in normal media are fated to form the lesion cap. Once the Notch3 program is turned on, cells are locked into a fibrous cap fate and do not give rise to osteochondrogenic SMCs. Importantly, Notch3 CreERT2 mice can be used as a cap-specific genetic manipulation tool.

Abstract 1043: Single-cell Characterization Of Mouse Aorta During Atherosclerosis Regression

Atherosclerosis is a cardiovascular disease characterized by the accumulation of fibro-fatty plaque in the arteries. Over half the cells in a mature atherosclerotic lesion are smooth muscle cells (SMCs) or SMC-derived cells (SDCs). SMCs can undergo a process called phenotypic switching, which involves dedifferentiation to an intermediate multipotent cell state, migration, and transdifferentiation into alternative cell types (SDCs). Increased SMC phenotypic switching is associated with worse atherosclerosis progression. However, little is known of the roles of SMCs and SDCs in atherosclerosis regression. We hypothesize that SDCs may promote plaque stability during atherosclerosis regression. In this study, we used an SMC lineage tracing mouse model (Ldlr–/–; LoxP-stop-LoxP-ZsGreen1+/-; Myh11-CreERT2+/-) that is limited to male mice. At 6 weeks of age, the mice were administered a tamoxifen diet for 1 week, followed by a week of chow diet. Mice were then fed a 16-week Western diet to induce atherosclerosis. We modeled lipid-lowering induced regression by switching to chow diet and administering HDAd-hLDLR. Mice were harvested at timepoints 0, 2, 5, 10, and 20 weeks. Arterial tissues, such as the aorta, brachiocephalic artery, and aortic root were used for histological characterization of disease and for single cell RNA-sequencing (scRNA-seq). The scRNA-seq data showed no major changes in SMC and SDC cell clusters, showing that our hypothesis was likely null. We noted an overall decrease in macrophages, and an increase in both of the two fibroblast clusters, which also had transcriptional signatures of increased proliferation and reduced apoptosis and senescence. Our histology data showed thicker fibrous caps and increased collagen, as expected in a regression model. Going forward, we will assess whether one or both fibroblast clusters are necessary for the stabilization of plaques during atherosclerosis regression.

Tracing embryonic hematopoiesis guides induction of pluripotent stem cells to hematopoietic progenitors

In this issue of Developmental Cell, Fowler etal. applied genetic lineage-tracing mouse models to support the notion that artery endothelial cells are the predominant source of hematopoietic stem cells. They leveraged this and developed a method capable of efficiently differentiating human pluripotent stem cells into HLF+HOXA+ hematopoietic progenitors.

From Hematopoietic Stem Cells to Platelets: Unifying Differentiation Pathways Identified by Lineage Tracing Mouse Models.

Platelets are the terminal progeny of megakaryocytes, primarily produced in the bone marrow, and play critical roles in blood homeostasis, clotting, and wound healing. Traditionally, megakaryocytes and platelets are thought to arise from multipotent hematopoietic stem cells (HSCs) via multiple discrete progenitor populations with successive, lineage-restricting differentiation steps. However, this view has recently been challenged by studies suggesting that (1) some HSC clones are biased and/or restricted to the platelet lineage, (2) not all platelet generation follows the "canonical" megakaryocytic differentiation path of hematopoiesis, and (3) platelet output is the default program of steady-state hematopoiesis. Here, we specifically investigate the evidence that in vivo lineage tracing studies provide for the route(s) of platelet generation and investigate the involvement of various intermediate progenitor cell populations. We further identify the challenges that need to be overcome that are required to determine the presence, role, and kinetics of these possible alternate pathways.

Mechanosensitive protein polycystin-1 promotes periosteal stem/progenitor cells osteochondral differentiation in fracture healing.

Background: Mechanical forces are indispensable for bone healing, disruption of which is recognized as a contributing cause to nonunion or delayed union. However, the underlying mechanism of mechanical regulation of fracture healing is elusive. Methods: We used the lineage-tracing mouse model, conditional knockout depletion mouse model, hindlimb unloading model and single-cell RNA sequencing to analyze the crucial roles of mechanosensitive protein polycystin-1 (PC1, Pkd1) promotes periosteal stem/progenitor cells (PSPCs) osteochondral differentiation in fracture healing. Results: Our results showed that cathepsin (Ctsk)-positive PSPCs are fracture-responsive and mechanosensitive and can differentiate into osteoblasts and chondrocytes during fracture repair. We found that polycystin-1 declines markedly in PSPCs with mechanical unloading while increasing in response to mechanical stimulus. Mice with conditional depletion of Pkd1 in Ctsk+ PSPCs show impaired osteochondrogenesis, reduced cortical bone formation, delayed fracture healing, and diminished responsiveness to mechanical unloading. Mechanistically, PC1 facilitates nuclear translocation of transcriptional coactivator TAZ via PC1 C-terminal tail cleavage, enhancing osteochondral differentiation potential of PSPCs. Pharmacological intervention of the PC1-TAZ axis and promotion of TAZ nuclear translocation using Zinc01442821 enhances fracture healing and alleviates delayed union or nonunion induced by mechanical unloading. Conclusion: Our study reveals that Ctsk+ PSPCs within the callus can sense mechanical forces through the PC1-TAZ axis, targeting which represents great therapeutic potential for delayed fracture union or nonunion.

Efficient Treatment of Pulpitis via Transplantation of Human Pluripotent Stem Cell-Derived Pericytes Partially through LTBP1-Mediated T Cell Suppression.

Dental pulp pericytes are reported to have the capacity to generate odontoblasts and express multiple cytokines and chemokines that regulate the local immune microenvironment, thus participating in the repair of dental pulp injury in vivo. However, it has not yet been reported whether the transplantation of exogenous pericytes can effectively treat pulpitis, and the underlying molecular mechanism remains unknown. In this study, using a lineage-tracing mouse model, we showed that most dental pulp pericytes are derived from cranial neural crest. Then, we demonstrated that the ablation of pericytes could induce a pulpitis-like phenotype in uninfected dental pulp in mice, and we showed that the significant loss of pericytes occurs during pupal inflammation, implying that the transplantation of pericytes may help to restore dental pulp homeostasis during pulpitis. Subsequently, we successfully generated pericytes with immunomodulatory activity from human pluripotent stem cells through the intermediate stage of the cranial neural crest with a high level of efficiency. Most strikingly, for the first time we showed that, compared with the untreated pulpitis group, the transplantation of hPSC-derived pericytes could substantially inhibit vascular permeability (the extravascular deposition of fibrinogen, ** p < 0.01), alleviate pulpal inflammation (TCR+ cell infiltration, * p < 0.05), and promote the regeneration of dentin (** p < 0.01) in the mouse model of pulpitis. In addition, we discovered that the knockdown of latent transforming growth factor beta binding protein 1 (LTBP1) remarkably suppressed the immunoregulation ability of pericytes in vitro and compromised their in vivo regenerative potential in pulpitis. These results indicate that the transplantation of pericytes could efficiently rescue the aberrant phenotype of pulpal inflammation, which may be partially due to LTBP1-mediated T cell suppression.

Extracellular Matrix Disorganization Caused by ADAMTS16 Deficiency Leads to Bicuspid Aortic Valve With Raphe Formation.

A better understanding of the molecular mechanism of aortic valve development and bicuspid aortic valve (BAV) formation would significantly improve and optimize the therapeutic strategy for BAV treatment. Over the past decade, the genes involved in aortic valve development and BAV formation have been increasingly recognized. On the other hand, ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) gene family members have been reported to be able to modulate cardiovascular development and diseases. The present study aimed to further investigate the roles of ADAMTS family members in aortic valve development and BAV formation. Morpholino-based ADAMTS family gene-targeted screening for zebrafish heart outflow tract phenotypes combined with DNA sequencing in a 304 cohort BAV patient registry study was initially carried out to identify potentially related genes. Both ADAMTS gene-specific fluorescence in situ hybridization assay and genetic tracing experiments were performed to evaluate the expression pattern in the aortic valve. Accordingly, related genetic mouse models (both knockout and knockin) were generated using the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9) method to further study the roles of ADAMTS family genes. The lineage-tracing technique was used again to evaluate how the cellular activity of specific progenitor cells was regulated by ADAMTS genes. Bulk RNA sequencing was used to investigate the signaling pathways involved. Inducible pluripotent stem cells derived from both BAV patients and genetic mouse tissue were used to study the molecular mechanism of ADAMTS. Immunohistochemistry was performed to examine the phenotype of cardiac valve anomalies, especially in the extracellular matrix components. ADAMTS genes targeting and phenotype screening in zebrafish and targeted DNA sequencing on a cohort of patients with BAV identified ADAMTS16 (a disintegrin and metalloproteinase with thrombospondin motifs 16) as a BAV-causing gene and found the ADAMTS16 p. H357Q variant in an inherited BAV family. Both in situ hybridization and genetic tracing studies described a unique spatiotemporal pattern of ADAMTS16 expression during aortic valve development. Adamts16+/- and Adamts16+/H355Q mouse models both exhibited a right coronary cusp-noncoronary cusp fusion-type BAV phenotype, with progressive aortic valve thickening associated with raphe formation (fusion of the commissure). Further, ADAMTS16 deficiency in Tie2 lineage cells recapitulated the BAV phenotype. This was confirmed in lineage-tracing mouse models in which Adamts16 deficiency affected endothelial and second heart field cells, not the neural crest cells. Accordingly, the changes were mainly detected in the noncoronary and right coronary leaflets. Bulk RNA sequencing using inducible pluripotent stem cells-derived endothelial cells and genetic mouse embryonic heart tissue unveiled enhanced FAK (focal adhesion kinase) signaling, which was accompanied by elevated fibronectin levels. Both in vitro inducible pluripotent stem cells-derived endothelial cells culture and ex vivo embryonic outflow tract explant studies validated the altered FAK signaling. Our present study identified a novel BAV-causing ADAMTS16 p. H357Q variant. ADAMTS16 deficiency led to BAV formation.

Fullerol rescues the light-induced retinal damage by modulating Müller glia cell fate

Excessive light exposure can damage photoreceptors and lead to blindness. Oxidative stress serves a key role in photo-induced retinal damage. Free radical scavengers have been proven to protect against photo-damaged retinal degeneration. Fullerol, a potent antioxidant, has the potential to protect against ultraviolet-B (UVB)-induced cornea injury by activating the endogenous stem cells. However, its effects on cell fate determination of Müller glia (MG) between gliosis and de-differentiation remain unclear. Therefore, we established a MG lineage-tracing mouse model of light-induced retinal damage to examine the therapeutic effects of fullerol. Fullerol exhibited superior protection against light-induced retinal injury compared to glutathione (GSH) and reduced oxidative stress levels, inhibited gliosis by suppressing the TGF-β pathway, and enhanced the de-differentiation of MG cells. RNA sequencing revealed that transcription candidate pathways, including Nrf2 and Wnt10a pathways, were involved in fullerol-induced neuroprotection. Fullerol-mediated transcriptional changes were validated by qPCR, Western blotting, and immunostaining using mouse retinas and human-derived Müller cell lines MIO-M1 cells, confirming that fullerol possibly modulated the Nrf2, Wnt10a, and TGF-β pathways in MG, which suppressed gliosis and promoted the de-differentiation of MG in light-induced retinal degeneration, indicating its potential in treating retinal diseases.

Modulation of the canonical Wnt activity by androgen signaling in prostate epithelial basal stem cells.

Both the canonical Wnt and androgen receptor (AR) signaling pathways are important for prostate organogenesis and homeostasis. How they crosstalk to regulate prostate stem cell behaviors remains unclear. Here, we show in lineage-tracing mouse models that although Wnt is essential for basal stem cell multipotency, ectopic Wnt activity promotes basal cell over-proliferation and squamous phenotypes, which are counteracted by elevated levels of androgen. In prostate basal cell organoids, dihydrotestosterone (DHT) antagonizes R-spondin-stimulated growth in a concentration-dependent manner. DHT down-regulates the expressions of a Wnt reporter and target genes, and RNA sequencing (RNA-seq) analyses identify Wnt signaling as a key altered pathway. Mechanistically, DHT enhances AR and β-catenin protein binding, and CUT&RUN analyses reveal that ectopic AR sequesters β-catenin away from its Wnt-related cistrome. Our results suggest that an intermediate level of Wnt activity in prostate basal stem cells, achieved via AR-β-catenin interaction, is essential for normal prostate homeostasis.

Cardiac pericytes mediate the remodeling response to myocardial infarction.

Despite the prevalence of pericytes in the microvasculature of the heart, their role during ischemia-induced remodeling remains unclear. We used multiple lineage-tracing mouse models and found that pericytes migrated to the injury site and expressed profibrotic genes, coinciding with increased vessel leakage after myocardial infarction (MI). Single-cell RNA-Seq of cardiac pericytes at various time points after MI revealed the temporally regulated induction of genes related to vascular permeability, extracellular matrix production, basement membrane degradation, and TGF-β signaling. Deleting TGF-β receptor 1 in chondroitin sulfate proteoglycan 4-expressing (Cspg4-expressing) cells reduced fibrosis following MI, leading to a transient improvement in the cardiac ejection fraction. Furthermore, genetic ablation of Cspg4-expressing cells resulted in excessive vascular permeability, a decline in cardiac function, and increased mortality in the second week after MI. These data reveal an essential role for cardiac pericytes in the control of vascular homeostasis and the fibrotic response after acute ischemic injury, information that will help guide the development of novel strategies to preserve vascular integrity and attenuate pathological cardiac remodeling.

Multilineage contribution of CD34+ cells in cardiac remodeling after ischemia/reperfusion injury

The ambiguous results of multiple CD34+ cell-based therapeutic trials for patients with heart disease have halted the large-scale application of stem/progenitor cell treatment. This study aimed to delineate the biological functions of heterogenous CD34+ cell populations and investigate the net effect of CD34+ cell intervention on cardiac remodeling. We confirmed, by combining single-cell RNA sequencing on human and mouse ischemic hearts and an inducible Cd34 lineage-tracing mouse model, that Cd34+ cells mainly contributed to the commitment of mesenchymal cells, endothelial cells (ECs), and monocytes/macrophages during heart remodeling with distinct pathological functions. The Cd34+-lineage-activated mesenchymal cells were responsible for cardiac fibrosis, while CD34+Sca-1high was an active precursor and intercellular player that facilitated Cd34+-lineage angiogenic EC-induced postinjury vessel development. We found through bone marrow transplantation that bone marrow-derived CD34+ cells only accounted for inflammatory response. We confirmed using a Cd34-CreERT2; R26-DTA mouse model that the depletion of Cd34+ cells could alleviate the severity of ventricular fibrosis after ischemia/reperfusion (I/R) injury with improved cardiac function. This study provided a transcriptional and cellular landscape of CD34+ cells in normal and ischemic hearts and illustrated that the heterogeneous population of Cd34+ cell-derived cells served as crucial contributors to cardiac remodeling and function after the I/R injury, with their capacity to generate diverse cellular lineages.

Abstract 363: Resident Vascular Adventitial Progenitor Cells Of Smooth Muscle Cell-lineage Adopt Profibrotic Phenotype And Contribute To Cardiac Fibrosis.

Cardiac myofibroblasts are the major contributors to ECM deposition in pathological cardiovascular fibrosis, which is the characteristic feature of cardiovascular diseases. However, due to potential heterogeneity of myofibroblasts, the origin of these cells remains controversial. Using highly specific smooth muscle cell lineage-tracing mouse models, we discovered the smooth muscle cell origin of a subpopulation of resident vascular adventitial progenitor cells, defined by the expression of the stem cell marker Sca1 (AdvSca1-SM cells), which rapidly proliferate and adopt a myofibroblast phenotype in response to acute vascular injury. Further, we identified a specific gene signature of active hedgehog/Wnt/β-catenin/Klf4 signaling in AdvSca1-SM cells and validated a Gli1-CreERT2-ROSA26-YFP (Gli1) reporter mouse model to be a faithful lineage tracing system for AdvSca1-SM cells. However, the function of AdvSca1-SM cells in cardiac fibrosis is unknown. Using immunofluorescent staining and label-free second harmonic generation (SHG) imaging, we observed the expansion and migration of AdvSca1-SM cells in close association with perivascular and interstitial cardiac fibrosis in pressure overload-induced cardiac fibrosis in Gli1 reporter mice. By integrating single cell RNA sequencing (scRNA-seq) with spatial transcriptomics, we identified a AngII-driven spatiotemporal profibrotic differentiation trajectory of AdvSca1-SM cells in mouse cardiac tissue. The trajectory, characterized by loss of expression of stemness genes, such as Klf4 , the lncRNA, Meg3 , and up-regulation of myofibroblast genes, was recapitulated in human RNA-seq data and of translational significance. Connectivity map analysis of the scRNA-seq data identified statins as potential candidates for inhibition of the profibrotic transition of AdvSca1-SM cells. In agreement, simvastatin induced the expression of stemness genes and inhibited TGFβ-induced up-regulation of αSMA and down-regulation of KLF4 in cultured AdvSca1-SM cells. Lentiviral-mediated up-regulation of Meg3 also rescued TGFβ-induced suppression of stemness genes. Our findings support the further development of anti-fibrotic therapeutics targeting AdcSca1-SM cells.

Abstract 217: Protease Activated Receptor 2 Is Critical For Vascular Smooth Muscle Cell Transition To A Macrophage-like State

Objective: Recent lineage tracing studies have shown vascular smooth muscle cells (VSMCs) develop macrophage-like characteristics (MLCs) in late-stage atherosclerosis resulting in accelerated inflammation and disease severity. Our objective was to determine if the inflammatory receptor, protease activated receptor 2 (PAR2), accelerates VSMC-MLC in a mouse model of atherosclerosis. Methods and Results: Our lab demonstrated Par2 -/- mice have decreased atherosclerotic lesion area versus proficient controls via downregulation of VSMC migration and cytokine release. To examine the role of PAR2 in VSMC dedifferentiation, Par2 +/+ and Par2 -/- VSMCs were treated with 10 ug/mL methyl- β-cyclodextrin cholesterol for 72 hours. While Par2 +/+ VSMCs demonstrated a loss of VSMCs markers (α - actin and myosin heavy chain) and upregulated macrophage markers (CD68 and Mac-2), Par2 -/- VSMCs remained stable. Looking at macrophage characteristics, Par2 -/- VSMCs phagocytized significantly less latex beads than Par2 +/+ VSMCs. Analysis of a hybrid mouse diversity panel (HMDP), conducted on 101 strains of mice with induced atherosclerosis, determined PAR2 was significantly correlated with the MLC super-gene Krüppel-like factor 4 (KLF4), which was found to have lower genetic expression in Par2 deficient VSMCs. Previous studies have demonstrated Par2 mRNA is bound and stabilized by the RNA binding protein human antigen R (HuR). Our studies confirm these findings where Par2 -/- VSMCs have less HuR mRNA expression and activity, suggesting a role of HuR in PAR2 mRNA stability. Par2 flox/flox mice bred with transgelin (SM22-alpha) Cre and low-density lipoprotein receptor deficient mice l have been establishing in our lab to investigate the effects of cell-specific deletions of PAR2 in atherosclerosis. VSMC-specific Par2 deficiency blunts the formation of atherosclerosis versus the Cre + littermates. Conclusions: These results suggest that VSMC-specific PAR2 activation mediates the dedifferentiation of VSMCs via upregulation of KLF4 and subsequent binding by HuR, which stabilizes PAR2 mRNA and upregulates PAR2. Future studies will continue to study VSMC dedifferentiation utilizing tagged lineage-tracing mouse models and various in vitro techniques.

Abstract NG02: Ordered and selective patterns of cancer genome evolution revealed by lineage tracing of sporadic p53 loss

Abstract Inactivation of the tumor suppressor p53 is associated with molecular features such as genomic instability, polyploidy, and the emergence of heterogeneous tumors that acquire diverse copy number alterations (CNAs). Clinically, p53 loss is associated with aggressive disease, therapeutic resistance, and poor prognosis. However, precisely when specific molecular features are selected following p53 inactivation and their evolutionary contribution to the initiation and progression of heterogenous p53 mutant tumors, remains poorly understood. This is in part because cancer genomes are typically analyzed after tumor development and not as they transverse the benign to malignant spectrum. Here, we develop a mouse model of pancreatic ductal adenocarcinoma (PDAC) that reports sporadic p53 loss in vivo, irrespective of phenotype, enabling the identification, isolation and lineage tracing of incipient cancer cells as they evolve through the benign to malignant transition. Using functional, histopathological, and genomics-based approaches, we find that cells undergoing p53 loss gradually gain malignant potential as they acquire CNAs in highly ordered and selective patterns. Initially, distinct p53-deficient clones emerge and compete via the selective acquisition of recurrent chromosomal deletions in otherwise diploid cells. Following the accumulation of many genomic deletions in a diploid state, a subset of highly rearranged clones undergo genome doubling, entering polyploid, presumably an adaptation to excess loss of gene dosage. In a polyploid state, rampant genomic diversification ensues involving the acquisition of additional deletions and, now, widespread chromosomal gains and amplifications resulting in a heterogenous malignant tumor. Despite vast heterogeneity, chromosomal deletions are selectively fixed as truncal, homogenous events in the tumor hierarchy. Retrospective analysis of whole genome, targeted-panel, and single-cell sequencing data of human PDAC corroborate these patterns in human disease. p53 was coined “guardian of the genome” 30 years ago upon the discovery that its inactivation prevents proliferative arrest following DNA damage, leading to genomic instability. However, the evolutionary relationship between p53 inactivation, associated genomic features, and the emergence of p53 tumors remains poorly understood. Leveraging a unique lineage tracing mouse model, we find that p53 inactivation results in a set of predictable evolutionary rules that are highly ordered and deterministic. These specific rules suggest stage dependent contributions of CNA events to malignant transformation and point to new strategies to target lethal, p53-deficient tumors. Citation Format: Timour Baslan. Ordered and selective patterns of cancer genome evolution revealed by lineage tracing of sporadic p53 loss. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr NG02.

Origins of pathological myofibroblasts in lung fibrosis: insights from lineage tracing mouse models in the single-cell RNA sequencing era.

Idiopathic pulmonary fibrosis (IPF) is a rare interstitial lung disease with a poor prognosis. Chronic microinjuries, mainly caused by environmental factors to an aging alveolar epithelium, would lead to the aberrant differentiation and accumulation of aberrant mesenchymal cells with a contractile phenotype, known as fibrosis-associated myofibroblasts, which trigger abnormal extracellular matrix accumulation and fibrosis. The origin of those pathological myofibroblasts in pulmonary fibrosis is not fully understood to date. Lineage tracing methods using mouse models have opened new avenues for studying cell fate in a pathological context. This review aims to present a nonexhaustive list of different potential sources of those harmful myofibroblasts during lung fibrosis, based on these in vivo approaches, and considering the normal and fibrotic lung cellular atlas recently established by single-cell RNA sequencing.