Tumor Biomechanics Alters Metastatic Dissemination of Triple Negative Breast Cancer via Rewiring Fatty Acid Metabolism.

In recent decades, the role of tumor biomechanics on cancer cell behavior at the primary site has been increasingly appreciated. However, the effect of primary tumor biomechanics on the latter stages of the metastatic cascade, such as metastatic seeding of secondary sites and outgrowth remains underappreciated. This work sought to address this in the context of triple negative breast cancer (TNBC), a cancer type known to aggressively disseminate at all stages of disease progression. Using mechanically tuneable model systems, mimicking the range of stiffness's typically found within breast tumors, it is found that, contrary to expectations, cancer cells exposed to softer microenvironments are more able to colonize secondary tissues. It is shown that heightened cell survival is driven by enhanced metabolism of fatty acids within TNBC cells exposed to softer microenvironments. It is demonstrated that uncoupling cellular mechanosensing through integrin ß1 blocking antibody effectively causes stiff primed TNBC cells to behave like their soft counterparts, both in vitro and in vivo. This work is the first to show that softer tumor microenvironments may be contributing to changes in disease outcome by imprinting on TNBC cells a greater metabolic flexibility and conferring discrete cell survival advantages.

Single-cell transcriptome dynamics of the autotaxin-lysophosphatidic acid axis during muscle regeneration reveal proliferative effects in mesenchymal fibro-adipogenic progenitors.

Lysophosphatidic acid is a growth factor-like bioactive phospholipid recognising LPA receptors and mediating signalling pathways that regulate embryonic development, wound healing, carcinogenesis, and fibrosis, via effects on cell migration, proliferation and differentiation. Extracellular LPA is generated from lysophospholipids by the secreted hydrolase-ectonucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2; also, AUTOTAXIN/ATX) and metabolised by different membrane-bound phospholipid phosphatases (PLPPs). Here, we use public bulk and single-cell RNA sequencing datasets to explore the expression of Lpar 1-6, Enpp2, and Plpp genes under skeletal muscle homeostasis and regeneration conditions. We show that the skeletal muscle system dynamically expresses the Enpp2-Lpar-Plpp gene axis, with Lpar1 being the highest expressed member among LPARs. Lpar1 was expressed by mesenchymal fibro-adipogenic progenitors and tenocytes, whereas FAPs mainly expressed Enpp2. Clustering of FAPs identified populations representing distinct cell states with robust Lpar1 and Enpp2 transcriptome signatures in homeostatic cells expressing higher levels of markers Dpp4 and Hsd11b1. However, tissue injury induced transient repression of Lpar genes and Enpp2. The role of LPA in modulating the fate and differentiation of tissue-resident FAPs has not yet been explored. Ex vivo, LPAR1/3 and ENPP2 inhibition significantly decreased the cell-cycle activity of FAPs and impaired fibro-adipogenic differentiation, implicating LPA signalling in the modulation of the proliferative and differentiative fate of FAPs. Together, our results demonstrate the importance of the ENPP2-LPAR-PLPP axis in different muscle cell types and FAP lineage populations in homeostasis and injury, paving the way for further research on the role of this signalling pathway in skeletal muscle homeostasis and regeneration, and that of other organs and tissues, in vivo.

PI(4,5)P2-dependent regulation of endothelial tip cell specification contributes to angiogenesis.

Dynamic positioning of endothelial tip and stalk cells, via the interplay between VEGFR2 and NOTCH signaling, is essential for angiogenesis. VEGFR2 activates PI3K, which phosphorylates PI(4,5)P2 to PI(3,4,5)P3, activating AKT; however, PI3K/AKT does not direct tip cell specification. We report that PI(4,5)P2 hydrolysis by the phosphoinositide-5-phosphatase, INPP5K, contributes to angiogenesis. INPP5K ablation disrupted tip cell specification and impaired embryonic angiogenesis associated with enhanced DLL4/NOTCH signaling. INPP5K degraded a pool of PI(4,5)P2 generated by PIP5K1C phosphorylation of PI(4)P in endothelial cells. INPP5K ablation increased PI(4,5)P2, thereby releasing ß-catenin from the plasma membrane, and concurrently increased PI(3,4,5)P3-dependent AKT activation, conditions that licensed DLL4/NOTCH transcription. Suppression of PI(4,5)P2 in INPP5K-siRNA cells by PIP5K1C-siRNA, restored ß-catenin membrane localization and normalized AKT signaling. Pharmacological NOTCH or AKT inhibition in vivo or genetic ß-catenin attenuation rescued angiogenesis defects in INPP5K-null mice. Therefore, PI(4,5)P2 is critical for ß-catenin/DLL4/NOTCH signaling, which governs tip cell specification during angiogenesis.

Mimicry of embryonic circulation enhances the hoxa hemogenic niche and human blood development.

Precursors of the adult hematopoietic system arise from the aorta-gonad-mesonephros (AGM) region shortly after the embryonic circulation is established. Here, we develop a microfluidic culture system to mimic the primitive embryonic circulation and address the hypothesis that circulatory flow and shear stress enhance embryonic blood development. Embryonic (HOXA+) hematopoiesis was derived from human pluripotent stem cells and induced from mesoderm by small-molecule manipulation of TGF-ß and WNT signaling (SB/CHIR). Microfluidic and orbital culture promoted the formation of proliferative CD34+RUNX1C-GFP+SOX17-mCHERRY+ precursor cells that were released into the artificial circulation from SOX17+ arterial-like structures. Single-cell transcriptomic analysis delineated extra-embryonic (yolk sac) and HOXA+ embryonic blood differentiation pathways. SB/CHIR and circulatory flow enhance hematopoiesis by the formation of proliferative HOXA+RUNX1C+CD34+ precursor cells that differentiate into monocyte/macrophage, granulocyte, erythrocyte, and megakaryocyte progenitors.

Tissue-Resident PDGFRα+ Progenitor Cells Contribute to Fibrosis versus Healing in a Context- and Spatiotemporally Dependent Manner.

PDGFRα+ mesenchymal progenitor cells are associated with pathological fibro-adipogenic processes. Conversely, a beneficial role for these cells during homeostasis or in response to revascularization and regeneration stimuli is suggested, but remains to be defined. We studied the molecular profile and function of PDGFRα+ cells in order to understand the mechanisms underlying their role in fibrosis versus regeneration. We show that PDGFRα+ cells are essential for tissue revascularization and restructuring through injury-stimulated remodeling of stromal and vascular components, context-dependent clonal expansion, and ultimate removal of pro-fibrotic PDGFRα+-derived cells. Tissue ischemia modulates the PDGFRα+ phenotype toward cells capable of remodeling the extracellular matrix and inducing cell-cell and cell-matrix adhesion, likely favoring tissue repair. Conversely, pathological healing occurs if PDGFRα+-derived cells persist as terminally differentiated mesenchymal cells. These studies support a context-dependent "yin-yang" biology of tissue-resident mesenchymal progenitor cells, which possess an innate ability to limit injury expansion while also promoting fibrosis in an unfavorable environment.

Platelet-derived growth factor-AB improves scar mechanics and vascularity after myocardial infarction.

Therapies that target scar formation after myocardial infarction (MI) could prevent ensuing heart failure or death from ventricular arrhythmias. We have previously shown that recombinant human platelet-derived growth factor-AB (rhPDGF-AB) improves cardiac function in a rodent model of MI. To progress clinical translation, we evaluated rhPDGF-AB treatment in a clinically relevant porcine model of myocardial ischemia-reperfusion. Thirty-six pigs were randomized to sham procedure or balloon occlusion of the proximal left anterior descending coronary artery with 7-day intravenous infusion of rhPDGF-AB or vehicle. One month after MI, rhPDGF-AB improved survival by 40% compared with vehicle, and cardiac magnetic resonance imaging showed left ventricular (LV) ejection fraction improved by 11.5%, driven by reduced LV end-systolic volumes. Pressure volume loop analyses revealed improved myocardial contractility and energetics after rhPDGF-AB treatment with minimal effect on ventricular compliance. rhPDGF-AB enhanced angiogenesis and increased scar anisotropy (high fiber alignment) without affecting overall scar size or stiffness. rhPDGF-AB reduced inducible ventricular tachycardia by decreasing heterogeneity of the ventricular scar that provides a substrate for reentrant circuits. In summary, we demonstrated that rhPDGF-AB promotes post-MI cardiac wound repair by altering the mechanics of the infarct scar, resulting in robust cardiac functional improvement, decreased ventricular arrhythmias, and improved survival. Our findings suggest a strong translational potential for rhPDGF-AB as an adjunct to current MI treatment and possibly to modulate scar in other organs.

Single-cell expression profiling reveals dynamic flux of cardiac stromal, vascular and immune cells in health and injury.

Besides cardiomyocytes (CM), the heart contains numerous interstitial cell types which play key roles in heart repair, regeneration and disease, including fibroblast, vascular and immune cells. However, a comprehensive understanding of this interactive cell community is lacking. We performed single-cell RNA-sequencing of the total non-CM fraction and enriched (Pdgfra-GFP+) fibroblast lineage cells from murine hearts at days 3 and 7 post-sham or myocardial infarction (MI) surgery. Clustering of >30,000 single cells identified >30 populations representing nine cell lineages, including a previously undescribed fibroblast lineage trajectory present in both sham and MI hearts leading to a uniquely activated cell state defined in part by a strong anti-WNT transcriptome signature. We also uncovered novel myofibroblast subtypes expressing either pro-fibrotic or anti-fibrotic signatures. Our data highlight non-linear dynamics in myeloid and fibroblast lineages after cardiac injury, and provide an entry point for deeper analysis of cardiac homeostasis, inflammation, fibrosis, repair and regeneration.

Intravital Imaging to Monitor Therapeutic Response in Moving Hypoxic Regions Resistant to PI3K Pathway Targeting in Pancreatic Cancer.

Application of advanced intravital imaging facilitates dynamic monitoring of pathway activity upon therapeutic inhibition. Here, we assess resistance to therapeutic inhibition of the PI3K pathway within the hypoxic microenvironment of pancreatic ductal adenocarcinoma (PDAC) and identify a phenomenon whereby pronounced hypoxia-induced resistance is observed for three clinically relevant inhibitors. To address this clinical problem, we have mapped tumor hypoxia by both immunofluorescence and phosphorescence lifetime imaging of oxygen-sensitive nanoparticles and demonstrate that these hypoxic regions move transiently around the tumor. To overlay this microenvironmental information with drug response, we applied a FRET biosensor for Akt activity, which is a key effector of the PI3K pathway. Performing dual intravital imaging of drug response in different tumor compartments, we demonstrate an improved drug response to a combination therapy using the dual mTORC1/2 inhibitor AZD2014 with the hypoxia-activated pro-drug TH-302.

Genetic burden and associations with adverse neurodevelopment in neonates with congenital heart disease.

BACKGROUND: Up to 20% of children with congenital heart disease (CHD) undergoing cardiac surgery develop neurodevelopmental disabilities (NDD), with some studies reporting persistent impairment. Recent large-scale studies have demonstrated shared genetic mechanisms contributing to CHD and NDD. In this study, a targeted approach was applied to assess direct clinical applicability of this information. METHODS: A gene panel comprising 148 known CHD and/or NDD genes was used to sequence 15 patients with CHD + NDD, 15 patients with CHD, and 15 healthy controls. The number and types of variants between the 3 groups were compared using Poisson log-linear regression, and the SNP-set (Sequence) Kernel Association Test-Optimized was used to conduct single-gene and gene-pathway burden analyses. RESULTS: A significant increase in rare (minor allele frequency < 0.01) and novel variants was identified between the CHD + NDD cohort and controls, P < .001 and P = .001, respectively. There was also a significant increase in rare variants in the CHD cohort compared with controls (P = .04). Rare variant burden analyses implicated pathways associated with "neurotransmitters," "axon guidance," and those incorporating "RASopathy" genes in the development of NDD in CHD patients. CONCLUSIONS: These findings suggest that an increase in novel and rare variants in known CHD and/or NDD genes is associated with the development of NDD in patients with CHD. Furthermore, burden analyses point toward rare variant burden specifically in pathways related to brain development and function as contributors to NDD. Although promising variants and pathways were identified, further research, utilizing whole-genome approaches, is required prior to demonstrating clinical utility in this patient group.

Control of cardiac jelly dynamics by NOTCH1 and NRG1 defines the building plan for trabeculation.

In vertebrate hearts, the ventricular trabecular myocardium develops as a sponge-like network of cardiomyocytes that is critical for contraction and conduction, ventricular septation, papillary muscle formation and wall thickening through the process of compaction 1 . Defective trabeculation leads to embryonic lethality2-4 or non-compaction cardiomyopathy (NCC) 5 . There are divergent views on when and how trabeculation is initiated in different species. In zebrafish, trabecular cardiomyocytes extrude from compact myocardium 6 , whereas in chicks, chamber wall thickening occurs before overt trabeculation 7 . In mice, the onset of trabeculation has not been described, but is proposed to begin at embryonic day 9.0, when cardiomyocytes form radially oriented ribs 2 . Endocardium-myocardium communication is essential for trabeculation, and numerous signalling pathways have been identified, including Notch2,8 and Neuregulin (NRG) 4 . Late disruption of the Notch pathway causes NCC 5 . Whereas it has been shown that mutations in the extracellular matrix (ECM) genes Has2 and Vcan prevent the formation of trabeculae in mice9,10 and the matrix metalloprotease ADAMTS1 promotes trabecular termination 3 , the pathways involved in ECM dynamics and the molecular regulation of trabeculation during its early phases remain unexplored. Here we present a model of trabeculation in mice that integrates dynamic endocardial and myocardial cell behaviours and ECM remodelling, and reveal new epistatic relationships between the involved signalling pathways. NOTCH1 signalling promotes ECM degradation during the formation of endocardial projections that are critical for individualization of trabecular units, whereas NRG1 promotes myocardial ECM synthesis, which is necessary for trabecular rearrangement and growth. These systems interconnect through NRG1 control of Vegfa, but act antagonistically to establish trabecular architecture. These insights enabled the prediction of persistent ECM and cardiomyocyte growth in a mouse NCC model, providing new insights into the pathophysiology of congenital heart disease.

A RhoA-FRET Biosensor Mouse for Intravital Imaging in Normal Tissue Homeostasis and Disease Contexts.

The small GTPase RhoA is involved in a variety of fundamental processes in normal tissue. Spatiotemporal control of RhoA is thought to govern mechanosensing, growth, and motility of cells, while its deregulation is associated with disease development. Here, we describe the generation of a RhoA-fluorescence resonance energy transfer (FRET) biosensor mouse and its utility for monitoring real-time activity of RhoA in a variety of native tissues in vivo. We assess changes in RhoA activity during mechanosensing of osteocytes within the bone and during neutrophil migration. We also demonstrate spatiotemporal order of RhoA activity within crypt cells of the small intestine and during different stages of mammary gestation. Subsequently, we reveal co-option of RhoA activity in both invasive breast and pancreatic cancers, and we assess drug targeting in these disease settings, illustrating the potential for utilizing this mouse to study RhoA activity in vivo in real time.

Transient tissue priming via ROCK inhibition uncouples pancreatic cancer progression, sensitivity to chemotherapy, and metastasis.

The emerging standard of care for patients with inoperable pancreatic cancer is a combination of cytotoxic drugs gemcitabine and Abraxane, but patient response remains moderate. Pancreatic cancer development and metastasis occur in complex settings, with reciprocal feedback from microenvironmental cues influencing both disease progression and drug response. Little is known about how sequential dual targeting of tumor tissue tension and vasculature before chemotherapy can affect tumor response. We used intravital imaging to assess how transient manipulation of the tumor tissue, or "priming," using the pharmaceutical Rho kinase inhibitor Fasudil affects response to chemotherapy. Intravital Förster resonance energy transfer imaging of a cyclin-dependent kinase 1 biosensor to monitor the efficacy of cytotoxic drugs revealed that priming improves pancreatic cancer response to gemcitabine/Abraxane at both primary and secondary sites. Transient priming also sensitized cells to shear stress and impaired colonization efficiency and fibrotic niche remodeling within the liver, three important features of cancer spread. Last, we demonstrate a graded response to priming in stratified patient-derived tumors, indicating that fine-tuned tissue manipulation before chemotherapy may offer opportunities in both primary and metastatic targeting of pancreatic cancer.

Dose Assessment and Considerations When a Radioactive Seed is Unrecoverable in a Breast Surgical Patient.

Excision of non-palpable breast tumors in cancer patients has been routinely performed using surgical clips and wires placed under mammographic or ultrasound guidance. An alternative method exists for performing these surgeries without surgical wires and this approach has become more widely used at Oncology Centers in the United States. Roswell Park Cancer Institute (RPCI) performs many of their breast surgeries with radioactive seeds and has enjoyed favorable outcomes along with an enhanced patient experience. There are some inherent difficulties with using radioactive seeds, and seed tracking can be a problem during surgical procedures. When a seed is misplaced, an extensive search is conducted in an effort to locate the misplaced seed. Every attempt to recover these radioactive seeds should be made unless patient safety is jeopardized. If the seed is unrecoverable, then a dose assessment must be performed and corrective action determined. It is important to have adequate policies and procedures in place to prevent these rare occurrences. They should be treated as a radioactive medical event.

A Universal and Robust Integrated Platform for the Scalable Production of Human Cardiomyocytes From Pluripotent Stem Cells.

UNLABELLED: Recent advances in the generation of cardiomyocytes (CMs) from human pluripotent stem cells (hPSCs), in conjunction with the promising outcomes from preclinical and clinical studies, have raised new hopes for cardiac cell therapy. We report the development of a scalable, robust, and integrated differentiation platform for large-scale production of hPSC-CM aggregates in a stirred suspension bioreactor as a single-unit operation. Precise modulation of the differentiation process by small molecule activation of WNT signaling, followed by inactivation of transforming growth factor-ß and WNT signaling and activation of sonic hedgehog signaling in hPSCs as size-controlled aggregates led to the generation of approximately 100% beating CM spheroids containing virtually pure (∼90%) CMs in 10 days. Moreover, the developed differentiation strategy was universal, as demonstrated by testing multiple hPSC lines (5 human embryonic stem cell and 4 human inducible PSC lines) without cell sorting or selection. The produced hPSC-CMs successfully expressed canonical lineage-specific markers and showed high functionality, as demonstrated by microelectrode array and electrophysiology tests. This robust and universal platform could become a valuable tool for the mass production of functional hPSC-CMs as a prerequisite for realizing their promising potential for therapeutic and industrial applications, including drug discovery and toxicity assays. SIGNIFICANCE: Recent advances in the generation of cardiomyocytes (CMs) from human pluripotent stem cells (hPSCs) and the development of novel cell therapy strategies using hPSC-CMs (e.g., cardiac patches) in conjunction with promising preclinical and clinical studies, have raised new hopes for patients with end-stage cardiovascular disease, which remains the leading cause of morbidity and mortality globally. In this study, a simplified, scalable, robust, and integrated differentiation platform was developed to generate clinical grade hPSC-CMs as cell aggregates under chemically defined culture conditions. This approach resulted in approximately 100% beating CM spheroids with virtually pure (∼90%) functional cardiomyocytes in 10 days from multiple hPSC lines. This universal and robust bioprocessing platform can provide sufficient numbers of hPSC-CMs for companies developing regenerative medicine technologies to rescue, replace, and help repair damaged heart tissues and for pharmaceutical companies developing advanced biologics and drugs for regeneration of lost heart tissue using high-throughput technologies. It is believed that this technology can expedite clinical progress in these areas to achieve a meaningful impact on improving clinical outcomes, cost of care, and quality of life for those patients disabled and experiencing heart disease.

ERBB2 triggers mammalian heart regeneration by promoting cardiomyocyte dedifferentiation and proliferation.

The murine neonatal heart can regenerate after injury through cardiomyocyte (CM) proliferation, although this capacity markedly diminishes after the first week of life. Neuregulin-1 (NRG1) administration has been proposed as a strategy to promote cardiac regeneration. Here, using loss- and gain-of-function genetic tools, we explore the role of the NRG1 co-receptor ERBB2 in cardiac regeneration. NRG1-induced CM proliferation diminished one week after birth owing to a reduction in ERBB2 expression. CM-specific Erbb2 knockout revealed that ERBB2 is required for CM proliferation at embryonic/neonatal stages. Induction of a constitutively active ERBB2 (caERBB2) in neonatal, juvenile and adult CMs resulted in cardiomegaly, characterized by extensive CM hypertrophy, dedifferentiation and proliferation, differentially mediated by ERK, AKT and GSK3ß/ß-catenin signalling pathways. Transient induction of caERBB2 following myocardial infarction triggered CM dedifferentiation and proliferation followed by redifferentiation and regeneration. Thus, ERBB2 is both necessary for CM proliferation and sufficient to reactivate postnatal CM proliferative and regenerative potentials.

Antisense-mediated exon skipping: a therapeutic strategy for titin-based dilated cardiomyopathy.

Frameshift mutations in the TTN gene encoding titin are a major cause for inherited forms of dilated cardiomyopathy (DCM), a heart disease characterized by ventricular dilatation, systolic dysfunction, and progressive heart failure. To date, there are no specific treatment options for DCM patients but heart transplantation. Here, we show the beneficial potential of reframing titin transcripts by antisense oligonucleotide (AON)-mediated exon skipping in human and murine models of DCM carrying a previously identified autosomal-dominant frameshift mutation in titin exon 326. Correction of TTN reading frame in patient-specific cardiomyocytes derived from induced pluripotent stem cells rescued defective myofibril assembly and stability and normalized the sarcomeric protein expression. AON treatment in Ttn knock-in mice improved sarcomere formation and contractile performance in homozygous embryos and prevented the development of the DCM phenotype in heterozygous animals. These results demonstrate that disruption of the titin reading frame due to a truncating DCM mutation can be restored by exon skipping in both patient cardiomyocytes in vitro and mouse heart in vivo, indicating RNA-based strategies as a potential treatment option for DCM.

An approach to radiation safety department benchmarking in academic and medical facilities.

Based on anecdotal evidence and networking with colleagues at other facilities, it has become evident that some radiation safety departments are not adequately staffed and radiation safety professionals need to increase their staffing levels. Discussions with management regarding radiation safety department staffing often lead to similar conclusions. Management acknowledges the Radiation Safety Officer (RSO) or Director of Radiation Safety's concern but asks the RSO to provide benchmarking and justification for additional full-time equivalents (FTEs). The RSO must determine a method to benchmark and justify additional staffing needs while struggling to maintain a safe and compliant radiation safety program. Benchmarking and justification are extremely important tools that are commonly used to demonstrate the need for increased staffing in other disciplines and are tools that can be used by radiation safety professionals. Parameters that most RSOs would expect to be positive predictors of radiation safety staff size generally are and can be emphasized in benchmarking and justification report summaries. Facilities with large radiation safety departments tend to have large numbers of authorized users, be broad-scope programs, be subject to increased controls regulations, have large clinical operations, have significant numbers of academic radiation-producing machines, and have laser safety responsibilities.

Cardiogenic genes expressed in cardiac fibroblasts contribute to heart development and repair.

RATIONALE: Cardiac fibroblasts are critical to proper heart function through multiple interactions with the myocardial compartment, but appreciation of their contribution has suffered from incomplete characterization and lack of cell-specific markers. OBJECTIVE: To generate an unbiased comparative gene expression profile of the cardiac fibroblast pool, identify and characterize the role of key genes in cardiac fibroblast function, and determine their contribution to myocardial development and regeneration. METHODS AND RESULTS: High-throughput cell surface and intracellular profiling of cardiac and tail fibroblasts identified canonical mesenchymal stem cell and a surprising number of cardiogenic genes, some expressed at higher levels than in whole heart. While genetically marked fibroblasts contributed heterogeneously to interstitial but not cardiomyocyte compartments in infarcted hearts, fibroblast-restricted depletion of one highly expressed cardiogenic marker, T-box 20, caused marked myocardial dysmorphology and perturbations in scar formation on myocardial infarction. CONCLUSIONS: The surprising transcriptional identity of cardiac fibroblasts, the adoption of cardiogenic gene programs, and direct contribution to cardiac development and repair provoke alternative interpretations for studies on more specialized cardiac progenitors, offering a novel perspective for reinterpreting cardiac regenerative therapies.

Nkx2-5(+)islet1(+) mesenchymal precursors generate distinct spleen stromal cell subsets and participate in restoring stromal network integrity.

Secondary lymphoid organ stromal cells comprise different subsets whose origins remain unknown. Herein, we exploit a genetic lineage-tracing approach to show that splenic fibroblastic reticular cells (FRCs), follicular dendritic cells (FDCs), marginal reticular cells (MRCs), and mural cells, but not endothelial cells, originate from embryonic mesenchymal progenitors of the Nkx2-5(+)Islet1(+) lineage. This lineage include embryonic mesenchymal cells with lymphoid tissue organizer (LTo) activity capable also of supporting ectopic lymphoid-like structures and a subset of resident spleen stromal cells that proliferate and regenerate the splenic stromal microenvironment following resolution of a viral infection. These findings identify progenitor cells that generate stromal diversity in spleen development and repair and suggest the existence of multipotent stromal progenitors in the adult spleen with regenerative capacity.

Selective inhibition of human group IIA-secreted phospholipase A2 (hGIIA) signaling reveals arachidonic acid metabolism is associated with colocalization of hGIIA to vimentin in rheumatoid synoviocytes.

Human group IIA secreted phospholipase A2 (hGIIA) promotes tumor growth and inflammation and can act independently of its well described catalytic lipase activity via an alternative poorly understood signaling pathway. With six chemically diverse inhibitors we show that it is possible to selectively inhibit hGIIA signaling over catalysis, and x-ray crystal structures illustrate that signaling involves a pharmacologically distinct surface to the catalytic site. We demonstrate in rheumatoid fibroblast-like synoviocytes that non-catalytic signaling is associated with rapid internalization of the enzyme and colocalization with vimentin. Trafficking of exogenous hGIIA was monitored with immunofluorescence studies, which revealed that vimentin localization is disrupted by inhibitors of signaling that belong to a rare class of small molecule inhibitors that modulate protein-protein interactions. This study provides structural and pharmacological evidence for an association between vimentin, hGIIA, and arachidonic acid metabolism in synovial inflammation, avenues for selective interrogation of hGIIA signaling, and new strategies for therapeutic hGIIA inhibitor design.