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Overexpression of Pim-1 and Pim-3 is associated with acquired resistance to MET inhibitors in MKN45 cells.

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Mesenchymal-epithelial transition (MET) blockade offers a new targeted therapy particularly in those cancers with MET amplification. However, the efficacy and the duration of the response to MET inhibitors are limited by the emergence of drug resistance. Here, we report that resistance to small-molecule inhibitors of MET can arise from increased expression of the prosurvival Pim protein kinases. This resistance mechanism was documented in non–small cell lung cancer and gastric cancer cells with MET amplification. Inhibition of Pim kinases enhanced cell death triggered by short-term treatment with MET inhibitors. Pim kinases control the translation of antiapoptotic protein Bcl-2 at an internal ribosome entry site and this mechanism was identified as the basis for Pim-mediated resistance to MET inhibitors. Protein synthesis was increased in drug-resistant cells, secondary to a Pim-mediated increase in cap-independent translation. In cells rendered drug resistant by chronic treatment with MET inhibitors, genetic or pharmacologic inhibition of Pim kinases was sufficient to restore sensitivity in vitro and in vivo. Taken together, our results rationalize Pim inhibition as a strategy to augment responses and blunt acquired resistance to MET inhibitors in cancer. Cancer Res; 75(24); 5318–28. ©2015 AACR.

Antidepressants, specifically Selective Serotonin Re-uptake Inhibitors (SSRIs), that alter serotonin metabolism are currently the most commonly prescribed drugs for the treatment of depression. There is some evidence to suggest these drugs contribute to birth defects. As jaw development is often altered in craniofacial birth defects, the purpose of this study was to interrogate the effects of in utero SSRI exposure in a preclinical model of mandible development.Wild type C57BL6 mice were used to produce litters that were exposed in utero to an SSRI, Citalopram (500 μg/day). Murine mandibles from P15 pups were analyzed for a change in shape and composition.Analysis indicated an overall shape change with total mandibular length and ramus height being shorter in exposed pups as compared to controls. Histomorphometric analysis revealed that first molar length was longer in exposed pups while third molar length was shorter in exposed as compared to control. Histological investigation of molars and surrounding periodontium revealed no change in collagen content of the molar in exposed pups, some alteration in collagen composition in the periodontium, increased alkaline phosphatase in molars and periodontium, and decreased mesenchymal cell marker presence in exposed mandibles.The results of this study reveal SSRI exposure may interrupt mandible growth as well as overall dental maturation in a model of development giving insight into the expectation that children exposed to SSRIs may require orthodontic intervention.

In human heart failure and in murine hearts with left-ventricular pressure overload (LVPO), increases in fibrosis are associated with increases in myocardial stiffness. Secreted protein acidic and rich in cysteine (SPARC) is shown to be necessary for both cardiac fibrosis and increases in myocardial stiffness in response to LVPO; however, cellular sources of cardiac SPARC are incompletely defined. Irradiation and bone marrow transfer were undertaken to test the hypothesis that SPARC expression by bone marrow-derived cells is an important mediator of fibrosis in LVPO. In recipient SPARC-null mice transplanted with donor wild-type (WT) bone marrow and subjected to LVPO, levels of fibrosis similar to that of WT mice were found despite the lack of SPARC expression by resident cells. In recipient WT mice with donor SPARC-null bone marrow, significantly less fibrosis versus that of WT mice was found despite the expression of SPARC by resident cells. Increases in myocardial stiffness followed a similar pattern to that of collagen deposition. Myocardial macrophages were significantly reduced in SPARC-null mice with LVPO versus that of WT mice. Recipient SPARC-null mice transplanted with donor WT bone marrow exhibited an increase in cardiac macrophages versus that of SPARC-null LVPO and donor WT mice with recipient SPARC-null bone marrow. Expression of vascular cellular adhesion molecule (VCAM), a previously identified binding partner of SPARC, was assessed in all groups and with the exception of WT mice, increases in VCAM immunoreactivity with LVPO were observed. However, no differences in VCAM expression between bone marrow transplant groups were noted. In conclusion, SPARC expression by bone marrow-derived cells was critical for fibrotic deposition of collagen and influenced the expansion of myocardial macrophages in response to LVPO. NEW & NOTEWORTHY Myocardial fibrosis and the resultant increases in LV and myocardial stiffness represent pivotal consequences of chronic pressure overload (PO). In this study, a murine model of cardiac fibrosis induced by PO was used to demonstrate a critical function of SPARC in bone marrow-derived cells that drives cardiac fibrosis and increases in cardiac macrophages.

Objective: The lungs are uniquely exposed to the external environment. Sand and dust exposures in desert regions are common among deployed soldiers. A significant number of Veterans deployed to the Middle East report development of respiratory disorders and diseases.Materials and methods: Sand collected from Fallujah, Iraq and Kandahar, Afghanistan combat zones was analyzed and compared to a sand sample collected from an historic United States (U.S.) battle region (Fort Johnson, James Island, SC, Civil War battle site). Sand samples were analyzed to determine the physical and elemental characteristics that may have the potential to contribute to development of respiratory disease.Results: Using complementary scanning electron microscopy (SEM) imaging and analysis, and inductively coupled plasma mass spectrometry (ICP-MS), it was determined that Iraq sand contained elevated levels of calcium and first row transition metals versus Afghanistan and U.S. sand. Iraq sand particle texture was smooth and round, and particles were considerably smaller than Afghanistan sand. Afghanistan sand was elevated in rare earth metals versus Iraq or U.S. sands and had sharp edge features and larger particle size than Iraq sand.Conclusions: These data demonstrate significant differences in Iraq and Afghanistan sand particle size and characteristics. Middle East sands contained elevated levels of elements that have been associated with respiratory disease versus control site sand, suggesting the potential of sand/dust storm exposure to promote adverse respiratory symptoms. Data also demonstrate the potential for variation based on geographical region or site of exposure. The data generated provide baseline information that will be valuable in designing future exposure studies.

Large bone injuries, defects, and chronic wounds present a major problem for medicine. Several therapeutic strategies are used clinically to precipitate bone including a combination therapy delivering osteoinductive bone morphogenetic protein 2 (rhBMP-2) via an osteoconductive scaffold (absorbable collagen sponge, ACS) (i.e. INFUSE). Adverse side effects reportedly associated with rhBMP2 administration include rampant inflammation and clinical failures. Although acute inflammation is necessary for proper healing in bone, inflammatory cascade dysregulation can result in sustained tissue damage and poor healing. We hypothesized that a subclinical dose of rhBMP2 modeled in the murine calvarial defect would not precipitate alterations to inflammatory markers during acute phases of bone wound healing. We utilized the 5mm critical size calvarial defect in C57BL6 wild-type mice which were subsequently treated with ACS and a subclinical dose of rhBMP2 shown to be optimal for healing. Three and seven-day post-operative time points were used to assess the role that rhBMP-2 plays in modulating inflammation versus ACS alone by cytokine array and histological interrogation. Data revealed that rhBMP-2 delivery resulted in substantial modulation of several markers associated with inflammation, most of which decreased to levels similar to control by the seven day time point. Additionally, while rhBMP-2 administration increased macrophage response, this peptide had little noticeable effect on traditional markers of macrophage polarization (M1-iNOS, M2-Arg1). These results suggest that rhBMP-2 delivered at a lower dose does not precipitate rampant inflammation. Thus, an assessment of dosing for rhBMP-2 therapies may lead to better healing outcomes and less surgical failure.

Neurological diseases, particularly in the context of aging, have serious impacts on quality of life and can negatively affect bone health. The brain-bone axis is critically important for skeletal metabolism, sensory innervation, and endocrine cross-talk between these organs. This review discusses current evidence for the cellular and molecular mechanisms by which various neurological disease categories, including autoimmune, developmental, dementia-related, movement, neuromuscular, stroke, trauma, and psychological, impart changes in bone homeostasis and mass, as well as fracture risk. Likewise, how bone may affect neurological function is discussed. Gaining a better understanding of brain-bone interactions, particularly in patients with underlying neurological disorders, may lead to development of novel therapies and discovery of shared risk factors, as well as highlight the need for broad, whole-health clinical approaches toward treatment.

Mechanisms that contribute to myocardial fibrosis, particularly in response to left ventricular pressure overload (LVPO), remain poorly defined. To test the hypothesis that a myocardial-specific profile of secreted factors is produced in response to PO, levels of 44 factors implicated in immune cell recruitment and function were assessed in a murine model of cardiac hypertrophy and compared with levels produced in a model of pulmonary fibrosis (PF). Mice subjected to PO were assessed at 1 and 4 wk. Protein from plasma, LV, lungs, and kidneys were analyzed by specific protein array analysis in parallel with protein from mice subjected to silica-instilled PF. Of the 44 factors assessed, 13 proteins were elevated in 1-wk PO myocardium, whereas 18 proteins were found increased in fibrotic lung. Eight of those increased in 1-wk LVPO were not found to be increased in fibrotic lungs (CCL-11, CCL-12, CCL-17, CCL-19, CCL-21, CCL-22, IL-16, and VEGF). Additionally, six factors were increased in plasma of 1-wk LVPO in the absence of increases in myocardial levels. In contrast, in mice with PF, no factors were found increased in plasma that were not elevated in lung tissue. Of those factors increased at 1 wk, only TIMP-1 remained elevated at 4 wk of LVPO. Immunohistochemistry of myocardial vasculature at 1 and 4 wk revealed similar amounts of total vasculature; however, evidence of activated endothelium was observed at 1 wk and, to a lesser extent, at 4 wk LVPO. In conclusion, PO myocardium generated a unique signature of cytokine expression versus that of fibrotic lung.NEW & NOTEWORTHY Myocardial fibrosis and the resultant increases in myocardial stiffness represent pivotal consequences of chronic pressure overload (PO). In this study, cytokine profiles produced in a murine model of cardiac fibrosis induced by PO were compared with those produced in response to silica-induced lung fibrosis. A unique profile of cardiac tissue-specific and plasma-derived factors generated in response to PO are reported.

Aims Data suggest pharmacological treatment of depression with selective serotonin reuptake inhibitors (SSRI) may impair bone health. Our group has previously modeled compromised craniofacial healing after treatment with sertraline, a commonly prescribed SSRI, and hypothesized potential culprits: alterations in bone cells, collagen, and/or inflammation. Here we interrogate bone lineage cell alterations due to sertraline treatment as a potential cause of the noted compromised bone healing. Main methods Murine pre-osteoblast, pre-osteoclast, osteoblast, and osteoclast cells were treated with clinically relevant concentrations of the SSRI. Studies focused on serotonin pathway targets, cell viability, apoptosis, differentiation, and the osteoblast/osteoclast feedback loop. Key findings All cells studied express neurotransmitters (e.g. serotonin transporter, SLC6A4, SSRI target) and G-protein-coupled receptors associated with the serotonin pathway. Osteoclasts presented the greatest native expression of Slc6a4 with all cell types exhibiting decreases in Slc6a4 expression after SSRI treatment. Pre-osteoclasts exhibited alteration to their differentiation pathway after treatment. Pre-osteoblasts and osteoclasts showed reduced apoptosis after treatment but showed no significant differences in functional assays. RANKL:OPG mRNA and protein ratios were decreased in the osteoblast lineage. Osteoclast lineage cells treated with sertraline demonstrated diminished TRAP positive cells when pre-exposed to sertraline prior to RANKL-induced differentiation. Significance These data suggest osteoclasts are a likely target of bone homeostasis disruption due to sertraline treatment, most potently through the osteoblast/clast feedback loop.

Heart failure (HF) has traditionally been defined by symptoms of fluid accumulation and poor perfusion, but it is now recognized that specific HF classifications hold prognostic and therapeutic relevance. Specifically, HF with reduced ejection fraction is characterized by reduced left ventricular systolic pump function and dilation and HF with preserved ejection fraction is characterized primarily by abnormal left ventricular filling (diastolic failure) with relatively preserved left ventricular systolic function. These forms of HF are distributed equally among patients with HF and likely require distinctly different strategies to mitigate the morbidity, mortality, and medical resource utilization of this disease. In particular, HF is a significant medical issue within the US Department of Veterans Affairs (VA) hospital system and constitutes a major translational research priority for the VA. Because a common underpinning of both HF with reduced ejection fraction and HF with preserved ejection fraction seems to be changes in the structure and function of the myocardial extracellular matrix, a conference was convened sponsored by the VA, entitled, "Targeting Myocardial Fibrosis in Heart Failure" to explore the extracellular matrix as a potential therapeutic target and to propose specific research directions. The conference was conceptually framed around the hypothesis that although HF with reduced ejection fraction and HF with preserved ejection fraction clearly have distinct mechanisms, they may share modifiable pathways and biological mediators in common. Inflammation and extracellular matrix were identified as major converging themes. A summary of our discussion on unmet challenges and possible solutions to move the field forward, as well as recommendations for future research opportunities, are provided.

Background aims Previous studies identified a circulating human osteoblastic population that expressed osteocalcin (OCN), increased following fracture and pubertal growth, and formed mineralized colonies in vitro and bone in vivo. A subpopulation expressed CD34, a hematopoietic/endothelial marker. These findings led to our hypothesis that hematopoietic-derived CD34+OCN+ cells exist in the circulation of mice and are modulated after fracture. Methods Flow cytometry was used to identify CD34+OCN+ cells in male B6.SJL-PtprcaPepcb/BoyJ and Vav-Cre/mTmG (VavR) mice. Non-stabilized tibial fractures were created by three-point bend. Fractures were longitudinally imaged by micro-computed tomography, and immunofluorescent staining was used to evaluate CD34+OCN+ cells within fracture callus. AMD3100 (10 mg/kg) was injected subcutaneously for 3 days and the CD34+OCN+ population was evaluated by flow cytometry. Results Circulating CD34+OCN+ cells were identified in mice and confirmed to be of hematopoietic origin (CD45+; Vav1+) using two mouse models. Both circulating and bone marrow-derived CD34+OCN+ cells peaked three weeks post-non-stabilized tibial fracture, suggesting association with cartilage callus transition to bone and early mineralization. Co-expression of CD34 and OCN in the fracture callus at two weeks post-fracture was observed. By three weeks, there was 2.1-fold increase in number of CD34+OCN+ cells, and these were observed throughout the fracture callus. AMD3100 altered CD34+OCN+ cell levels in peripheral blood and bone marrow. Discussion Together, these data demonstrate a murine CD34+OCN+ circulating population that may be directly involved in fracture repair. Future studies will molecularly characterize CD34+OCN+ cells, determine mechanisms regulating their contribution, and examine if their number correlates with improved fracture healing outcomes.

Cellular immunity established via immunotherapy holds the potential to eliminate solid tumors. Yet, cancer vaccines have failed to induce tumor-reactive T cells of sufficient quality to control disease. The inducible T cell costimulator (ICOS) pathway has been implicated in both the selective induction of immunity over tolerance as well as licensing of IL-17-polarized cellular immunity. Herein, we evaluated the ability of ICOS ligand (ICOSL) to augment the immunogenicity of adenoviral-based vaccination targeting the unglycosylated MUC1 peptide antigen. Vaccination disrupted immunotolerance in a transgenic mouse model recognizing human MUC1 as a self-antigen, inducing robust MUC1-specific immunity. Augmenting vaccination with ICOSL induced a bipolar Th17/Th1 effector profile, marked by increased MUC1-specific IL-17A production and RORγt expression in CD4+ but not CD8+ T cells which predominantly expressed IFNγ/IL-2 and T-bet. The polarization and maintenance of Th17 cells established following ICOSL augmented vaccination was highly durable, with elevated IL-17A and RORγt levels detected in CD4+ T cells up to 10 months after initial immunization. Furthermore, provision of ICOSL significantly enhanced MUC1-specific IgG antibody in response to immunization. ICOSL signaling dramatically influenced CD4+ T cell phenotype, altering gene expression of transcription factors and regulators of effector function following immunization. Interestingly, ICOSL augmentation failed to alter the transcriptional profile of CD8+ T cells following immunization, affecting the magnitude, but not distribution, of gene expression. Collectively, ICOSL supports the induction of durable, antigen-specific Th17/Th1-mediated immunity in vivo, establishing a vaccination platform to enhance CD4+ T cell-mediated antitumor immunity and providing a crucial component of an effective cancer vaccine.

Large craniofacial defects present a substantial clinical challenge that often requires the use of osteoconductive matrices and osteoinductive cues (i.e., bone morphogenetic proteins [BMP2]) to augment healing. While these methods have improved clinical outcomes, a better understanding of how the osteogenic fronts surrounding the defect, the underlying dura mater, and the cranial suture area contribute to healing may lead to more targeted therapies to enhance bone regeneration. We hypothesized that healing within a large bone defect will be precipitated from cells within the remaining or available suture mesenchyme abutting the edges of a murine critical sized defect. To investigate this hypothesis, 39 adult, wild-type mice were randomly arranged into groups (9 or 10 per group) by time (4 and 8 weeks) and treatment (control, acellular collagen sponge alone, or acellular collagen sponge loaded with a clinically relevant scaled dosage of BMP2). The skulls were then subjected to microcomputed tomography and histological analysis to assess bone regeneration in regions of interest within the defect area. A regional assessment of healing indicated that BMP2 drives greater healing than control and that healing emanates from the surgical margin, particularly from the margin associated with undisrupted suture mesenchyme. Though BMP2 treatment drove an increase in cell presence within the healing defect, there was no regional orientation of craniofacial stem cells or vascularity. Overall, these data reinforce that osteoconductive matrices in conjunction with osteoinductive peptides result in better healing of large calvarial defects. This healing is characterized as emanating from the surgical margin where there is an abundant supply of vasculature and progenitor cells.

AIMS: Following an acute myocardial infarction (MI) the extracellular matrix (ECM) undergoes remodeling in order to prevent dilation of the infarct area and maintain cardiac output. Excessive and prolonged inflammation following an MI exacerbates adverse ventricular remodeling. Macrophages are an integral part of the inflammatory response that contribute to this remodeling. Treatment with histone deacetylase (HDAC) inhibitors preserves LV function and myocardial remodeling in the post-MI heart. This study tested whether inhibition of HDAC activity resulted in preserving post-MI LV function through the regulation of macrophage phenotype and early resolution of inflammation. METHODS AND RESULTS: HDAC inhibition does not affect the recruitment of CD45(+) leukocytes, CD45(+)/CD11b(+) inflammatory monocytes or CD45(+)/CD11b(+)CD86(+) inflammatory macrophages for the first 3 days following infarct. Further, HDAC inhibition does not change the high expression level of the inflammatory cytokines in the first days following MI. However, by day 7, there was a significant reduction in the levels of CD45(+)/Cd11b(+) and CD45(+)/CD11b(+)/CD86(+) cells with HDAC inhibition. Remarkably, HDAC inhibition resulted in the dramatic increase in the recruitment of CD45(+)/CD11b(+)/CD206(+) alternatively activated macrophages as early as 1 Day which remained significantly elevated until 5 days post-MI. qRT-PCR revealed that HDAC inhibitor treatment shifts the cytokine and chemokine environment towards an M2 phenotype with upregulation of M2 markers at 1 and 5 days post-MI. Importantly, HDAC inhibition correlates with significant preservation of both LV ejection fraction and end-diastolic volume and is associated with a significant increase in micro-vessel density in the border zone at 14 days post-MI. CONCLUSION: Inhibition of HDAC activity result in the early recruitment of reparative CD45(+)/CD11b(+)/CD206(+) macrophages in the post-MI heart and correlates with improved ventricular function and remodeling. This work identifies a very promising therapeutic opportunity to manage macrophage phenotype and enhance resolution of inflammation in the post-MI heart.

Hypertension (HTN) has long been associated with abdominal aortic aneurysm (AAA) development, and these cardiovascular pathologies are biochemically characterized by elevated plasma levels of angiotensin II (AngII) as well as interleukin-6 (IL-6). A biologic relationship between HTN and AAA has not been established, however. Accordingly, the objective of this study was to evaluate whether elevated tension may initiate IL-6 production to accumulate monocyte/macrophages and promote dilation of the abdominal aorta (AA).An IL-6 infusion model (4.36 μg/kg/day) was created utilizing an osmotic infusion pump, and after 4 weeks, AA diameter was measured by digital microscopy. The AA was then excised for CD68 immunostaining and flow cytometric analysis with CD11b and F4/80 to identify macrophages. Aortic segments from wild-type mice were suspended on parallel wires in an ex vivo tissue myograph at experimentally derived optimal tension (1.2 g) and in the presence of elevated tension (ET, 1.7 g) for 3 hr, and expression of IL-6 and monocyte chemoattractant protein-1 (MCP-1) was evaluated by quantitative polymerase chain reaction (QPCR). Isolated aortic vascular smooth muscle cells (VSMCs) were subjected to 12% biaxial cyclic stretch or held static (control) for 3 hr (n = 7), and IL-6 and MCP-1 expressions were evaluated by QPCR.Four-week IL-6 infusion resulted in an AA outer diameter that was 72.5 ± 5.6% (P 0.05) greater than that of control mice, and aortic dilation was accompanied by an accumulation of macrophages in the AA medial layer as defined by an increase in CD68 + staining as well as an increase by flow cytometric quantification of CD11b+/F4/80+ cells. Wild-type AA segments did not respond to ex vivo application of ET but cyclic stretch of isolated VSMCs increased IL-6 (2.03 ± 0.3 fold) and MCP-1 (1.51 ± 0.11 fold) expression compared to static control (P 0.05). Pretreatment with the selective STAT3 inhibitor WP1066 blunted the response in both cases. Interestingly, AngII did not stimulate expression of IL-6 and MCP-1 above that initiated by tension and again, the response was inhibited by WP1066, supporting an integral role of STAT3 in this pathway.An IL-6 infusion model can initiate macrophage accumulation as well as aortic dilation, and under conditions of elevated tension, this proinflammatory cytokine can be produced by aortic VSMCs. By activation of STAT3, MCP-1 is expressed to increase media macrophage abundance and create an environment susceptible to dilation. This biomechanical association between HTN and aortic dilation may allow for the identification of novel therapeutic strategies.

BackgroundTumor-associated macrophages (TAMs) exert profound influence over breast cancer progression, promoting immunosuppression, angiogenesis, and metastasis.1 Neuropilin-2 (NRP2), consisting of NRP2a and NRP2b isoforms, is a co-receptor for heparin-binding growth factors including VEGF-C and Class 3 Semaphorins. Selective upregulation in response to environmental stimuli and independent signaling pathways endow the NRP2 isoforms with unique functionality.2 3 We have shown that the two isoforms of NRP2 endow opposing functionality to tumor cells due to distinct signaling pathways, with NRP2b promoting metastatic behavior.3 Although NRPs have been shown to regulate macrophage/TAM biology, the role of NRP2 isoforms in TAM functionality has yet to be evaluated.MethodsTo assess the contribution of NRP2 isoforms to macrophage biology, conditional NRP2a and NRP2b knockout mice and stable shRNA knockdown of NRP2a or NRP2b in Raw264.7 macrophages were generated. Phagocytosis, lysosomal processing of phagocytosed cargo, cytokine production, and influence on tumor cell migration were assessed in vitro using NRP2 isoform knockdown macrophages. NRP2 isoform expression was evaluated on TAMs from murine 4T1 and EO771 mammary carcinoma models using spectral cytometry and single-cell qPCR. NRP2 isoforms and approximated immune composition were evaluated in paired primary tumors and distant metastasis using RNAseq in a cohort of 99 breast cancer patients. High-dimensional myeloid phenotyping was performed on malignant pleural effusions (MPEs) from breast cancer patients or effusions of benign origin using 33-color spectral cytometry and unbiased computational analysis.ResultsNRP2 isoform expression was significantly increased in TAMs from murine tumors compared to macrophages from healthy mammary glands. NRP2 isoforms in human primary and metastatic breast cancer were strongly correlated with one another and positively correlated with increased TAMs. Distinct phenotypes of NRP2 isoform-expressing TAMs in were present in 4T1 and EO771 mouse breast cancers and within MPEs from breast cancer patients which were associated with high levels of activation and potential response to a hypoxic tumor niche. Genetic depletion of either NRP2 isoform resulted in dramatic reduction of LPS-induced IL-10 production, defects in phagosomal processing of apoptotic breast cancer cells, and increase in cancer cell migration following co-culture. By contrast, inhibition of IL-6 production was specific in NRP2b knockdown cells while phagocytic uptake of labeled particulates was inhibited only by NPR2a knockdown.ConclusionsThese results demonstrate that NRP2 isoforms regulate both shared and distinct functionality in macrophages and that NRP2 isoform expression identifies unique TAM subsets in breast cancer.AcknowledgementsThis work was supported by awards from the Susan G. Komen Foundation (CCR15329745), U.S. Department of Defense (W81XWH1910650), and American Lung Association/Thoracic Surgery Foundation to ACS. RD was supported by funding from a Department of Veteran's Affairs Career Development Award (CX001771-01A2) and the University of Pittsburgh's Dean Faculty Advancement Award. ESY was supported by the NCI of the NIH under R03 CA245774. MTL was supported by the NCI of the NIH under awards R01CA181450 and R01CA206012 as well as ITTC/UPMCE. RD and ACS were further supported by funding from the Department of Cardiothoracic Surgery.ReferencesWilliams CB, Yeh ES, Soloff AC. Tumor-associated macrophages: unwitting accomplices in breast cancer malignancy. Npj Breast Cancer [Internet]. Breast Cancer Research Foundation/Macmillan Publishers Limited; 2016;2:15025. Available from: http://dx.doi.org/10.1038/npjbcancer.2015.252.Nasarre P, Gemmill RM, Potiron VA, Roche J, Lu X, Barón AE, et al. Neuropilin-2 is upregulated in lung cancer cells during TGF-β1–Induced epithelial–mesenchymal transition. Cancer Res [Internet] 2013;73:7111 LP–7121. Available from: http://cancerres.aacrjournals.org/content/73/23/7111.abstract3.Gemmill RM, Nasarre P, Nair-Menon J, Cappuzzo F, Landi L, D'Incecco A, et al. The neuropilin 2 isoform NRP2b uniquely supports TGFβ-mediated progression in lung cancer. Sci Signal [Internet] 2017;10. Available from: http://stke.sciencemag.org/content/10/462/eaag0528.abstractEthics ApprovalThe study was approved by the University of Pittsburgh's Institutional Review Board approval number CR19120172-005.

Aims Respiratory disorders are a prominent component of Gulf War Illness. Although much of the underlying mechanisms of Gulf War Illness remain undefined, chronic immune dysfunction is a consistent feature of this multi-symptomatic, multi-organ disorder. Alveolar macrophages represent the predominant mononuclear phagocytes of the pulmonary mucosa, orchestrating the host response to pathogens and environmental stimuli. Herein, we sought to characterize the innate immune response of the pulmonary mucosa, with a focus on macrophages, to experimental respiratory exposure to two putative Gulf War Toxins (GWTs). Materials and methods Utilizing commercially available instrumentation, we evaluated the effect of aerosolized exposure to the pesticide malathion and diesel exhaust particulate (DEP) on the immune composition and inflammatory response of the lung in FVB/N mice using multiparametric spectral cytometry, cytokine analysis, and histology. Key findings Aerosolized GWTs induced gross pulmonary pathology with transient recruitment of neutrophils and sustained accumulation of alveolar macrophages to the lung for up to two weeks after exposure cessation. High-dimensional cytometry and unbiased computational analysis identified novel myeloid subsets recruited to the lung post-exposure driven by an influx of peripheral monocyte-derived progenitors. DEP and malathion, either alone or in combination, induced soluble mediators in bronchoalveolar lavage indicative of oxidative stress (PGF2α), inflammation (LTB4, TNFα, IL-12), and immunosuppression (IL-10), that were sustained or increased two weeks after exposures concluded. Significance These findings indicate that macrophage accumulation and pulmonary inflammation induced by GWTs continue in the absence of toxin exposure and may contribute to the immunopathology of respiratory Gulf War Illness.

Although bone repair is typically an efficient process, an inadequate healing response can occur, with approximately 5-20% of fractures developing nonunion. Even with improved healing strategies and external fixation devices, overall rate of nonunion has not been significantly reduced, particularly for atrophic nonunion. Atrophic nonunion is characterized by sparse or no callus formation and is difficult to treat clinically, resulting in long-term pain and functional limitation. Reliable preclinical models are needed to study the pathophysiology of atrophic nonunion to create better treatment options. The MouseNail kit (RISystem, Landquart, Switzerland) provides a highly standardized approach in which stabilized segmental bone defects are achieved through interlocked intramedullary nailing. However, reliably performing this surgery is technically challenging, particularly while maintaining strict asepsis. Skilled and aseptic surgical execution is important and necessary because it ensures optimal animal welfare and reproducibility. Therefore, the aim of this paper is to describe:•Novel modifications to the MouseNail kit that allow for: 1) a completely aseptic surgical environment, including description of a hanging limb orthopedic aseptic preparation and 2) a reduction in fracture gap size necessary for induction of atrophic nonunion.•Pre- to post-operative recommendations to facilitate successful performance of murine orthopedic survival surgery., Graphical Abstract Image, graphical abstract

Currently, there is no cure for Osteogenesis imperfecta (OI) - a debilitating pediatric skeletal dysplasia. Herein we show that hematopoietic stem cell (HSC) therapy holds promise in treating OI. Using single cell HSC transplantation in lethally irradiated oim/oim mice, we demonstrate significant improvements in bone morphometric, mechanics and turnover parameters. Importantly, we highlight that HSCs cause these improvements due to their unique property of differentiating into osteoblasts/osteocytes, depositing healthy collagen- an attribute thus far assigned only to mesenchymal stem cells. To confirm this HSC plasticity, lineage tracing was done by transplanting oim/oim with HSCs from two specific transgenic mice - VavR, in which all hematopoietic cells are GFP + and pOBCol2.3GFP, where GFP is expressed only in osteoblasts/osteocytes. In both models, transplanted oim/oim mice demonstrated presence of GFP + HSC-derived osteoblasts/osteocytes in bones. These studies unequivocally establish that HSCs differentiate into osteoblasts/osteocytes and HSC transplantation can provide a new translational approach for OI.

The Centers for Disease Control and Prevention, National Birth Defects Study suggests that environmental exposures including maternal thyroid diseases, maternal nicotine use, and use of selective serotonin reuptake inhibitors (SSRIs) may exacerbate incidence and or severity of craniofacial abnormalities including craniosynostosis. Premature fusion of a suture(s) of the skull defines the birth defect craniosynostosis which occurs in 1:1800-2500 births. A proposed mechanism of craniosynostosis is the disruption of proliferation and differentiation of cells in the perisutural area. Here, we hypothesize that pharmacological exposures including excess thyroid hormone, nicotine, and SSRIs lead to an alteration of stem cells within the sutures resulting in premature fusion. In utero exposure to nicotine and citalopram (SSRI) increased the risk of premature suture fusion in a wild-type murine model. Gli1+ stem cells were reduced, stem cell populations were depleted, and homeostasis of the suture mesenchyme was altered with exposure. Thus, although these pharmacological exposures can deplete calvarial stem cell populations leading to craniosynostosis, depletion of stem cells is not a unifying mechanism for pharmacological exposure associated craniosynostosis. Keywords: Craniosynostosis, Stem cells, Gli1, Teratogens

The significant biochemical and physiological effects of psychological stress are beginning to be recognized as exacerbating common diseases, including osteoporosis. This review discusses the current evidence for psychological stress-associated mental health disorders as risk factors for osteoporosis, the mechanisms that may link these conditions, and potential implications for treatment. Traditional, alternative, and adjunctive therapies are discussed. This review is not intended to provide therapeutic recommendations, but, rather, the goal of this review is to delineate potential interactions of psychological stress and osteoporosis and to highlight potential multi-system implications of pharmacological interventions. Review of the current literature identifies several potentially overlapping mechanistic pathways that may be of interest (e.g., glucocorticoid signaling, insulin-like growth factor signaling, serotonin signaling) for further basic and clinical research. Current literature also supports the potential for cross-effects of therapeutics for osteoporosis and mental health disorders. While studies examining a direct link between osteoporosis and chronic psychological stress are limited, the studies reviewed herein suggest that a multi-factorial, personalized approach should be considered for improved patient outcomes in populations experiencing psychological stress, particularly those at high-risk for development of osteoporosis.

Aim: To evaluate the comparative risk of hip fracture or osteoporosis among US Veterans based on selective serotonin reuptake inhibitor (SSRI) exposure. Patients & methods: A retrospective cohort study of eligible US Veterans Health Administration patients enrolled in 2003–2004 was performed to examine SSRIs’ 2-, 5- and 10-year impact on bone health using multiple logistic regression. Results: Veterans on SSRIs were found to be 56.7% more likely over a 10-year period to suffer a hip fracture (risk ratio: 1.567; 95% CI: 1.464–1.676) and 34.6% more likely to develop osteoporosis (risk ratio: 1.346; 95% CI: 1.319–1.374) when compared with those who were SSRI naive. Conclusion: SSRI usage was associated with greater risk of hip fracture and osteoporosis over a 10-year period in the veteran population, with similar effect sizes to smaller studies.

Despite the link between adverse birth outcomes due to pre- and peri-natal nicotine exposure, research suggests 11% of US women continue to smoke or use alternative nicotine products throughout pregnancy. Maternal smoking has been linked to incidence of craniofacial anomalies. We hypothesized that pre-natal nicotine exposure may directly alter craniofacial development independent of the other effects of cigarette smoking. To test this hypothesis, we administered pregnant C57BL6 mice drinking water supplemented with 0, 50, 100 or 200 μg/ml nicotine throughout pregnancy. On postnatal day 15 pups were sacrificed and skulls underwent micro-computed tomography (µCT) and histological analyses. Specific nicotinic acetylcholine receptors, α3, α7, β2, β4 were identified within the calvarial growth sites (sutures) and centers (synchondroses). Exposing murine calvarial suture derived cells and isotype cells to relevant circulating nicotine levels alone and in combination with nicotinic receptor agonist and antagonists resulted in cell specific effects. Most notably, nicotine exposure increased proliferation in calvarial cells, an effect that was modified by receptor agonist and antagonist treatment. Currently it is unclear what component(s) of cigarette smoke is causative in birth defects, however these data indicate that nicotine alone is capable of disrupting growth and development of murine calvaria.

Respiratory disorders including chronic obstructive pulmonary disease, asthma, and increased susceptibility to lung infections are a prominent component of Gulf War Illness (GWI). Although the mechanisms of GWI are unknown, chronic immune dysfunction is a consistent factor of this multi-symptomatic disorder. Alveolar macrophages (AMs) represent the predominant mononuclear phagocytes of the pulmonary mucosa, orchestrating the host response to pathogens and environmental stimuli via balanced induction of inflammatory, anti-inflammatory, and pro-resolution mediators. Utilizing commercially available instrumentation, we evaluated the effect of aerosolized exposure to two putative GW toxins, the pesticide malathion and diesel exhaust particulate (DEP), on the immune composition and inflammatory response of the lung in FVB/N mice. Toxin exposure resulted in gross lung pathology with variable increases in protein, lactose dehydrogenase, and cytokines in bronchoalveolar lavage (BAL). Aerosolized toxin exposure reduced AMs in the lung and BAL, associated with a shift to wound-healing phenotypes and concomitant influx of monocyte-derived progenitors from the circulation. Exposure to DEP and malathion, either alone or in combination, induced varying levels of dysfunction in flow-sorted AMs ex vivo as determined by inhibited migration, cytokine production, and phagocytosis. Toxin-induced alterations were associated with unique transcriptional profiles following RNA sequencing analysis. Collectively, these findings suggest that respiratory toxin-induced macrophage dysfunction may establish a state of frustrated immune resolution underlying the chronic immunopathology of respiratory GWI.

The peripheral auditory nerve (AN) carries sound information from sensory hair cells to the brain. The present study investigated the contribution of mouse and human hematopoietic stem cells (HSCs) to cellular diversity in the AN following the destruction of neuron cell bodies, also known as spiral ganglion neurons (SGNs). Exposure of the adult mouse cochlea to ouabain selectively killed type I SGNs and disrupted the blood-labyrinth barrier. This procedure also resulted in the upregulation of genes associated with hematopoietic cell homing and differentiation, and provided an environment conducive to the tissue engraftment of circulating stem/progenitor cells into the AN. Experiments were performed using both a mouse-mouse bone marrow transplantation model and a severely immune-incompetent mouse model transplanted with human CD34+ cord blood cells. Quantitative immunohistochemical analysis of recipient mice demonstrated that ouabain injury promoted an increase in the number of both HSC-derived macrophages and HSC-derived nonmacrophages in the AN. Although rare, a few HSC-derived cells in the injured AN exhibited glial-like qualities. These results suggest that human hematopoietic cells participate in remodeling of the AN after neuron cell body loss and that hematopoietic cells can be an important resource for promoting AN repair/regeneration in the adult inner ear.

Advanced glycation end products, or AGEs, and their corresponding receptor, the receptor for AGE (RAGE), have roles in several chronic diseases such as diabetes and cardiovascular disease due to their proinflammatory properties. Recently AGE: RAGE signaling has been implicated in a number of cancers, including prostate and breast cancer; however, how AGE: RAGE signaling effects these cancers has not been elucidated. To examine this, we have developed a novel dietary AGE tumor model. Our regular and AGE (TestDiet DIO 58G7) diets consist of equal %k/cal from fat, protein, and carbohydrate; however, the predominant source of carbohydrate in the control diet is starch and in the AGE diet is sugar. To generate the high-AGE diet we autoclave at 120°C for 15 min. In the high-AGE diet, the autoclaving mimics the cooking process in foods and induces the formation of AGEs, allowing us to observe the effects of chronic AGE consumption on both normal developmental processes as well as tumor formation and progression. Specifically, we have been able to show the impact of this diet during time periods where the breast is more susceptible to chronic AGE consumption, namely the “windows of susceptibility.” Additionally, we have applied this novel dietary model to the RAGE null (-/-) mice to explore the role of AGE:RAGE signaling further. Specifically, we have shown that chronic consumption of a high-AGE diet disrupts normal pubertal growth in the mammary gland through the recruitment of aberrant stroma, including fibroblasts and macrophages, and that these aberrant changes persist into adulthood. We further show that dietary AGEs promote aggressive tumor growth through manipulation of the tumor microenvironment and that this is RAGE dependent. We propose that this dietary model can be used as an in vivo method to examine the impact of dietary AGEs on the TME and as a mechanism for potential therapeutic and preventative strategies. Citation Format: Reid K. Schuster, Bradley A. Krisanits, Jamie Randise, Lourdes M. Nogueira, Kristi Helke, Amanda C. LaRue, David P. Turner, Victoria J. Findlay. A novel dietary advanced glycation end product (AGE) model to assess impact on tumor progression [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr B26.

Introduction. The focus of this study is on early lifestyle factors and their effect on mammary development during puberty and how they relate to increased breast cancer risk and disparities. At this time we do not understand what biological changes occur during pubertal mammary development which leads to a greater risk of developing cancer in later life. Identifying the molecular mechanisms that cause aberrant pubertal mammary development may lead to defined strategies to reduce breast cancer burden in later life. As our bodies use the sugars that we consume for energy they generate waste chemicals known as advanced glycation end products or AGEs for short. Significantly, low income, obesity and a sedentary lifestyle are established factors driving health disparity that also contribute to increased AGE accumulation levels in our bodies. In particular, AGE content in the Western Diet has consistently increased over the last 50 years due to increased consumption of sugar-laden and cheap processed/manufactured foods which are high in reactive AGE metabolites and can promote obesity. Methods. We use a dietary mouse model to assess impact of AGE on normal mammary development. Wild type FVB/n and RAGE null (RAGE-/-) mice are fed the respective diets from weaning until 7 (pubertal) or 12 (adult) weeks of age. Mammary glands are extracted for whole mounting and paraffin embedded for histology. Fibroblasts were isolated from mammary glands and cultured ex vivo. Transwell migration assays were performed with isolated fibroblasts and HC11 mouse mammary epithelial cells. qPCR was performed on the isolated fibroblasts to assess their activation status. Results. Early life exposures during mammary development influence the breast microenvironment to increase breast cancer risk. We show that due to an innate ability to influence the cellular matrix, dietary AGEs disrupt developmental programs during puberty and promote breast tumor growth. Through receptor for AGE (RAGE) dependent and independent mechanisms, chronic AGE consumption delayed ductal extension, increased ductal branching and caused aberrant terminal end bud (TEB) morphology. Dietary AGE activation of RAGE mediated a program of activated stroma leading to hyperplastic growth and the formation of pre-neoplastic lesions which persisted into adulthood. Importantly, AGE mediated effects remained even after diet intervention after puberty. In dietary-AGE breast tumor models, AGE mediated changes in tissue architecture and cell function were recapitulated and resulted in 3-fold increase in neoplastic growth. Through the perpetual activation of a reactive stroma, AGEs derived from diet represent a common early life exposure which can influence tumor behavior. Conclusions. A greater mechanistic understanding of the link between AGE intake during puberty and increased breast cancer risk may define novel potential strategies for lifestyle and pharmacological intervention aimed at reducing breast cancer risk and cancer disparities. Citation Format: Callan C Frye, Bradley A Krisanits, Reid Schuster, Jaime Randise, Lourdes M Nogueira, Kristi Helke, Amanda C LaRue, David P Turner, Victoria J Findlay. Consumption of dietary AGEs during puberty and increased breast cancer risk: A link between lifestyle and cancer disparity [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr C029.

Chronic inflammation is evident in the adipose tissue and periphery of patients with obesity, as well as mouse models of obesity. T cell subsets in obese adipose tissue are skewed towards Th1- and Th17-associated phenotypes and their secreted cytokines contribute to obesity-associated inflammation. Our lab recently identified a novel, myeloid-derived CD45+DDR2+ cell subset that modulates T cell activity. The current study sought to determine how these myeloid-derived CD45+DDR2+ cells are altered in the adipose tissue and peripheral blood of preobese mice and how this population modulates T cell activity. C57BL/6 mice were fed with a diet high in milkfat (60%·kcal, HFD) ad libitum until a 20% increase in total body weight was reached, and myeloid-derived CD45+DDR2+ cells and CD4+ T cells in visceral adipose tissue (VAT), mammary gland-associated adipose tissue (MGAT), and peripheral blood (PB) were phenotypically analyzed. Also analyzed was whether mediators from MGAT-primed myeloid-derived CD45+DDR2+ cells stimulate normal CD4+ T cell cytokine production. A higher percentage of myeloid-derived CD45+DDR2+ cells expressed the activation markers MHC II and CD80 in both VAT and MGAT of preobese mice. CD4+ T cells were preferentially skewed towards Th1- and Th17-associated phenotypes in the adipose tissue and periphery of preobese mice. In vitro, MGAT from HFD-fed mice triggered myeloid-derived CD45+DDR2+ cells to induce CD4+ T cell IFN-γ and TNF-α production. Taken together, this study shows that myeloid-derived CD45+DDR2+ cells express markers of immune activation and suggests that they play an immune modulatory role in the adipose tissue of preobese mice.

The scaffold/adaptor growth factor receptor bound 2 (GRB2)-associated binding protein 2 (GAB2) is frequently amplified and/or overexpressed in primary high-grade serous ovarian cancers (HGSOCs). Here we investigate a novel treatment strategy by targeting SHP2 and PI3K signaling in HGSOCs with GAB2 amplification/overexpression (GAB2High). The expression of GAB2 was analyzed in primary HGSOCs and ovarian cancer cell lines. In vitro and in vivo assays were performed to demonstrate the effect of SHP2 and PI3K-mediated GAB2High HGSOC progression. Analysis of gene expression data reveals that primary GAB2High HGSOCs are associated with increased ERBB, RAS, and MAPK activity signatures. Inhibition of SHP2 by an allosteric inhibitor SHP099 selectively inhibits ERK1/2 activity, proliferation, and survival of GAB2High ovarian cancer cell lines. Treatment with SHP099 has a synergistic effect with BKM120, a pan-class I PI3K inhibitor, at suppressing proliferation and survival of GAB2High ovarian cancer cells in vitro and in vivo by more effectively activating both BIM and BAD and inhibiting c-MYC compared with individual inhibitor. Our findings identify an important role of SHP2 in promoting proliferation and survival of GAB2High ovarian cancer cells, and combinatorial SHP2 and PI3K inhibition may be a promising therapeutic approach for such cancer.

Chronic inflammation is evident in the adipose tissue and periphery of patients with obesity, as well as mouse models of obesity. T cell subsets in obese adipose tissue are skewed towards Th1- and Th17-associated phenotypes and their secreted cytokines contribute to obesity-associated inflammation. Our lab recently identified a novel, myeloid-derived CD45+DDR2+ cell subset that modulates T cell activity. The current study sought to determine how these myeloid-derived CD45+DDR2+ cells are altered in the adipose tissue and peripheral blood of preobese mice and how this population modulates T cell activity. C57BL/6 mice were fed with a diet high in milkfat (60%·kcal, HFD) ad libitum until a 20% increase in total body weight was reached, and myeloid-derived CD45+DDR2+ cells and CD4+ T cells in visceral adipose tissue (VAT), mammary gland-associated adipose tissue (MGAT), and peripheral blood (PB) were phenotypically analyzed. Also analyzed was whether mediators from MGAT-primed myeloid-derived CD45+DDR2+ cells stimulate normal CD4+ T cell cytokine production. A higher percentage of myeloid-derived CD45+DDR2+ cells expressed the activation markers MHC II and CD80 in both VAT and MGAT of preobese mice. CD4+ T cells were preferentially skewed towards Th1- and Th17-associated phenotypes in the adipose tissue and periphery of preobese mice. In vitro, MGAT from HFD-fed mice triggered myeloid-derived CD45+DDR2+ cells to induce CD4+ T cell IFN-γ and TNF-α production. Taken together, this study shows that myeloid-derived CD45+DDR2+ cells express markers of immune activation and suggests that they play an immune modulatory role in the adipose tissue of preobese mice.

Bone wound healing is a highly dynamic and precisely controlled process through which damaged bone undergoes repair and complete regeneration. External factors can alter this process, leading to delayed or failed bone wound healing. The findings of recent studies suggest that the use of selective serotonin reuptake inhibitors (SSRIs) can reduce bone mass, precipitate osteoporotic fractures and increase the rate of dental implant failure. With 10% of Americans prescribed antidepressants, the potential of SSRIs to impair bone healing may adversely affect millions of patients’ ability to heal after sustaining trauma. Here, we investigate the effect of the SSRI sertraline on bone healing through pre-treatment with (10 mg·kg-1 sertraline in drinking water, n = 26) or without (control, n = 30) SSRI followed by the creation of a 5-mm calvarial defect. Animals were randomized into three surgical groups: (a) empty/sham, (b) implanted with a DermaMatrix scaffold soak-loaded with sterile PBS or (c) DermaMatrix soak-loaded with 542.5 ng BMP2. SSRI exposure continued until sacrifice in the exposed groups at 4 weeks after surgery. Sertraline exposure resulted in decreased bone healing with significant decreases in trabecular thickness, trabecular number and osteoclast dysfunction while significantly increasing mature collagen fiber formation. These findings indicate that sertraline exposure can impair bone wound healing through disruption of bone repair and regeneration while promoting or defaulting to scar formation within the defect site.

Many variables serve to alter the process of bone remodelling and diminish regeneration including the size and nature of the wound bed and health status of the individual. To overcome these inhibitory factors, tissue-engineered osteoconductive scaffolds paired with various growth factors have been utilized clinically. However, many limitations still remain, for example, bone morphogenetic protein 2 (BMP2) can lead to rampant inflammation, ectopic bone formation, and graft failure. Here, we studied the ability for a nanofiber scaffold (Talymed) to accelerate BMP2 growth factor-induced bone healing compared with the traditional absorbable collagen sponge (ACS) delivery system. One hundred fifty-five adult wild type mice were arranged in 16 groups by time, 4 and 8 weeks, and treatment, ACS or Talymed, loaded with control, low, medium, or high dosages of BMP2. Skulls were subjected to microCT, biomechanical, and histological analysis to assess bone regeneration. The use of Talymed within the defect site was found to decrease the bone volume, bone formation rate, and alkaline phosphatase activity compared with ACS/BMP2 combinations. Interestingly, though Talymed regenerated less bone, the regenerate was found to have a greater hardness value than that of bone within the ACS groups. However, the difference in bone hardness between scaffolds was not detectable by 8 weeks. Based on these results, we found that the nanofiber scaffold generated a better quality of bone regenerate at 4 weeks but, due to the lack of overall bone formation and the inhibition of normal remodelling processes, was not as efficacious as the current clinical standard ACS/BMP2 therapy.

Myocardial fibrosis and the resultant increases in left ventricular stiffness represent pivotal consequences of chronic pressure overload (PO) that impact both functional capacity and the rates of morbid and mortal events. However, the time course and cellular mechanisms that underlie PO-induced fibrosis have not been completely defined. Secreted protein acidic and rich in cysteine (SPARC) is a matricellular protein that has been shown to be required for insoluble collagen deposition and increased myocardial stiffness in response to PO in mice. As macrophages are associated with increases in fibrillar collagen, the hypothesis that macrophages represent a source of increased SPARC production in the PO myocardium was tested. The time course of changes in the myocardial macrophage population was compared with changes in procollagen type I mRNA, production of SPARC, fibrillar collagen accumulation, and diastolic stiffness. In PO hearts, mRNA encoding collagen type I was increased at 3 days, whereas increases in levels of total collagen protein did not occur until 1 wk and were followed by increases in insoluble collagen at 2 wk. Increases in muscle stiffness were not detected before increases in insoluble collagen content (>1 wk). Significant increases in myocardial macrophages that coincided with increased SPARC were found but did not coincide with increases in mRNA encoding collagen type I. Furthermore, immunohistochemistry and flow cytometry identified macrophages as a cellular source of SPARC. We conclude that myocardial macrophages play an important role in the time-dependent increases in SPARC that enhance postsynthetic collagen processing, insoluble collagen content, and myocardial stiffness and contribute to the development of fibrosis. NEW & NOTEWORTHY Myocardial fibrosis and the resultant increases in left ventricular and myocardial stiffness represent pivotal consequences of chronic pressure overload. In this study a murine model of cardiac fibrosis induced by pressure overload was used to establish a time course of collagen expression, collagen deposition, and cardiac macrophage expansion.

Almost two decades ago, a number of cell culture and preclinical transplantation studies suggested the striking concept of the tissue-reconstituting ability of hematopoietic stem cells (HSCs). While this heralded an exciting time of radically new therapies for disorders of many organs and tissues, the concept was soon mired by controversy and remained dormant. This chapter provides a brief review of evidence for HSC plasticity including our findings based on single HSC transplantation in mouse. These studies strongly support the concept that HSCs are pluripotent and may be the source for the majority, if not all, of the cell types in our body.

While earlier studies have suggested that cells positive for hematopoietic markers can be found in dental tissues, it has yet to be confirmed. To conclusively demonstrate this, we utilized a unique transgenic model in which all hematopoietic cells are green fluorescent protein+ (GFP+). Pulp, periodontal ligament (PDL) and alveolar bone (AvB) cell culture analysis demonstrated numerous GFP+ cells, which were also CD45+ (indicating hematopoietic origin) and co-expressed markers of cellular populations in pulp (dentin matrix protein-1, dentin sialophosphoprotein, alpha smooth muscle actin [ASMA], osteocalcin), in PDL (periostin, ASMA, vimentin, osteocalcin) and in AvB (Runx-2, bone sialoprotein, alkaline phosphatase, osteocalcin). Transplantation of clonal population derived from a single GFP+ hematopoietic stem cell (HSC), into lethally irradiated recipient mice, demonstrated numerous GFP+ cells within dental tissues of recipient mice, which also stained for markers of cell populations in pulp, PDL and AvB (used above), indicating that transplanted HSCs can differentiate into cells in dental tissues. These hematopoietic-derived cells deposited collagen and can differentiate in osteogenic media, indicating that they are functional. Thus, our studies demonstrate, for the first time, that cells in pulp, PDL and AvB can have a hematopoietic origin, thereby opening new avenues of therapy for dental diseases and injuries.