First Annual PACT Act Research Symposium on Veterans Health: A Colorado PACT Act Collaboration (CoPAC) Initiative.

In response to the Sergeant First Class Heath Robinson Honoring our Promise to Address Comprehensive Toxics (PACT) Act being signed into law, several research groups in Colorado organized the First Annual PACT Act Research Symposium for Veteran Health. The 2-day symposium was interested in research relevant to military exposures with a primary focus on respiratory and mental health. Information on the PACT Act, data sources in the Department of Veteran Affairs and DOD, and research opportunities at the Veteran Affairs were presented. The morning session centered on respiratory health, highlighting research conducted over the last two decades regarding deployment-related respiratory diseases. Despite the high prevalence of mental health disorders among Veterans, information presented during the afternoon sessions on mental health highlighted the dearth of research to date regarding psychological health and military-related exposures. Policymakers, clinicians, and researchers were encouraged to adopt a life-course approach when conceptualizing physical and psychological exposures. On the second day of meetings, a smaller group of participants discussed next steps in military exposure research, as well as priorities for future research. Per the latter, recommendations for future research were made regarding the need for more precise exposure characterization, longitudinal data collection, and efforts to increase understanding regarding disease pathogenesis, as well as the impact of exposures across multiple organs. Such efforts will require interdisciplinary collaboration.

Application-specific approaches to MicroCT for evaluation of mouse models of pulmonary disease.

The advent of micro-computed tomography (microCT) has provided significant advancement in our ability to generate clinically relevant assessments of lung health and disease in small animal models. As microCT use to generate outcomes analysis in pulmonary preclinical models has increased there have been substantial improvements in image quality and resolution, and data analysis software. However, there are limited published methods for standardized imaging and automated analysis available for investigators. Manual quantitative analysis of microCT images is complicated by the presence of inflammation and parenchymal disease. To improve the efficiency and limit user-associated bias, we have developed an automated pulmonary air and tissue segmentation (PATS) task list to segment lung air volume and lung tissue volume for quantitative analysis. We demonstrate the effective use of the PATS task list using four distinct methods for imaging, 1) in vivo respiration controlled scanning using a flexiVent, 2) longitudinal breath-gated in vivo scanning in resolving and non-resolving pulmonary disease initiated by lipopolysaccharide-, bleomycin-, and silica-exposure, 3) post-mortem imaging, and 4) ex vivo high-resolution scanning. The accuracy of the PATS task list was compared to manual segmentation. The use of these imaging techniques and automated quantification methodology across multiple models of lung injury and fibrosis demonstrates the broad applicability and adaptability of microCT to various lung diseases and small animal models and presents a significant advance in efficiency and standardization of preclinical microCT imaging and analysis for the field of pulmonary research.

Evaluation of SARS-CoV-2 antibody point of care devices in the laboratory and clinical setting.

SARS-CoV-2 antibody tests have been marketed to diagnose previous SARS-CoV-2 infection and as a test of immune status. There is a lack of evidence on the performance and clinical utility of these tests. We aimed to carry out an evaluation of 14 point of care (POC) SARS-CoV-2 antibody tests. Serum from participants with previous RT-PCR (real-time polymerase chain reaction) confirmed SARS-CoV-2 infection and pre-pandemic serum controls were used to determine specificity and sensitivity of each POC device. Changes in sensitivity with increasing time from infection were determined on a cohort of study participants. Corresponding neutralising antibody status was measured to establish whether the detection of antibodies by the POC device correlated with immune status. Paired capillary and serum samples were collected to ascertain whether POC devices performed comparably on capillary samples. Sensitivity and specificity varied between the POC devices and in general did not meet the manufacturers' reported performance characteristics, which signifies the importance of independent evaluation of these tests. The sensitivity peaked at ≥20 days following onset of symptoms, however sensitivity of 3 of the POC devices evaluated at extended time points showed that sensitivity declined with time. This was particularly marked at >140 days post infection. This is relevant if the tests are to be used for sero-prevalence studies. Neutralising antibody data showed that positive antibody results on POC devices did not necessarily confer high neutralising antibody titres, and that these POC devices cannot be used to determine immune status to the SARS-CoV-2 virus. Comparison of paired serum and capillary results showed that there was a decline in sensitivity using capillary blood. This has implications in the utility of the tests as they are designed to be used on capillary blood by the general population.

Healing after COVID-19: are survivors at risk for pulmonary fibrosis?

The novel SARS-CoV-2 coronavirus, which is responsible for COVID-19 disease, was first reported in Wuhan, China, in December of 2019. The virus rapidly spread, and the World Health Organization declared a pandemic by March 2020. With millions of confirmed cases worldwide, there is growing concern and considerable debate regarding the potential for coronavirus infection to contribute to an appreciable burden of chronic respiratory symptoms or fibrotic disease among recovered individuals. Because the first case of COVID-19 was documented less than one year ago, data regarding long-term clinical outcomes are not yet available, and predictions for long-term outcome are speculative at best. However, due to the staggering number of cases and the severity of disease in many individuals, there is a critical need to consider the potential long-term implications of COVID-19. This review examines current basic and clinical data regarding fibrogenic mechanisms of viral injury in the context of SARS-CoV-2. Several intersecting mechanisms between coronavirus infection and fibrotic pathways are discussed to highlight factors and processes that may be targetable to improve patient outcome. Reports of post-infection sequelae from previous coronavirus outbreaks are presented toward the goal of improved recognition of potential contributing risk factors for fibrotic disease.

Comparison of SARS-CoV-2 serological assays for use in epidemiological surveillance in Scotland.

Background: Sero-surveillance of SARS-CoV-2 is crucial to monitoring levels of population exposure and informing public health responses, but may be influenced by variability in performance between available assays. Methods: Five commercial immunoassays and a neutralising activity assay were used to detect antibodies to SARS-CoV-2 in routine primary care and paediatric samples collected during the first wave of the pandemic in NHS Lothian, Scotland as part of ongoing surveillance efforts. For each assay, sensitivity and specificity was calculated relative to consensus results (majority of immunoassays positive = overall positive) and neutralising activity. Quantitative correlation was performed between serological and neutralising titres. Results: Seroprevalence ranged from 3.4-7.3 % in primary care patients and 3-5.9 % in paediatric patients according to different immunoassays. Neutralising activity was detectable in 2.8 % and 1.3 % respectively. Relative assay performance changed depending on comparison to immunoassay consensus versus neutralising activity and qualititative versus quantitative agreement. Cross-reactivity with endemic seasonal coronaviruses was confirmed by neutralising assay in false positives for one immunoassay. Presence of false positives for another assay was found specifically in paediatric but not adult samples. Conclusions: Five serological assays show variable accuracy when applied to the general population, impacting seroprevalence estimates. Assay performance may also vary in detection of protective neutralising antibody levels. These aspects should be considered in assay selection and interpretation in epidemiological studies.

SPARC production by bone marrow-derived cells contributes to myocardial fibrosis in pressure overload.

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.

Physical and elemental analysis of Middle East sands from recent combat zones.

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.

An atypical DYRK kinase connects quorum-sensing with posttranscriptional gene regulation in Trypanosoma brucei.

The sleeping sickness parasite, Trypanosoma brucei, uses quorum sensing (QS) to balance proliferation and transmission potential in the mammal bloodstream. A signal transduction cascade regulates this process, a component of which is a divergent member of the DYRK family of protein kinases, TbDYRK. Phylogenetic and mutational analysis in combination with activity and phenotypic assays revealed that TbDYRK exhibits a pre-activated conformation and an atypical HxY activation loop motif, unlike DYRK kinases in other eukaryotes. Phosphoproteomic comparison of TbDYRK null mutants with wild-type parasites identified molecules that operate on both the inhibitory 'slender retainer' and activatory 'stumpy inducer' arms of the QS control pathway. One of these molecules, the RNA-regulator TbZC3H20, regulates parasite QS, this being dependent on the integrity of its TbDYRK phosphorylation site. This analysis reveals fundamental differences to conventional DYRK family regulation and links trypanosome environmental sensing, signal transduction and developmental gene expression in a coherent pathway.

Examining outcomes following thrombolysis in an increasingly older and dependent stroke population.

BACKGROUND: Thrombolysis for acute ischaemic stroke (AIS) patients aged ˜80 years is evidence based, although its use in previously dependent patients is controversial. METHODS: Data from 831 thrombolysed AIS patients in our centre from 2009-2017 were used to compare demographic trends and outcomes (haemorrhage, mortality, three-month independence) in patients aged <80 and ˜80 years and with prior dependency. Comparison with UK and world registry data regarding age and pre-stroke dependency was made. RESULTS: The percentage of treated patients aged ˜80 years increased year-on-year, doubling from 25% to 50% (p <0.01), with increasing average age and pre-stroke dependency in world centres. Patients ˜80 years had higher (p <0.001) stroke severity, symptomatic intracerebral haemorrhage (5% vs. 1.5%), mortality (35% vs. 13%) and lower three month independent survival (24% vs. 60%). Patients with pre-stroke dependency had especially higher three month mortality (57-71%, OR 3.75 [95% CI 1.97-7.15]) in both age groups. CONCLUSION: Patients aged ˜80 years and with dependency increasingly receive thrombolysis. Given poorer outcomes thrombolysis trials are needed in pre-stroke dependent patients.

Early Blood Profile of C57BL/6 Mice Exposed to Chronic Unpredictable Stress.

Physiological responses to psychological stressors are protective in acute fight or flight situations; however, there is increasing evidence suggesting the detrimental impact of chronic psychological stress on disease. Chronic stress has been associated with inflammation, poor prognosis, increased morbidity, and poor outcome in many diseases including atherosclerosis, cancer, and pulmonary disease. Given the systemic impact of stress, and the role of the hematopoietic system as a rapid responder to homeostatic insults, we hypothesized that early blood profile changes and biochemical alterations could be detected in a model of chronic stress. To test this hypothesis, a variation of the chronic unpredictable stress (CUS) model was employed. Following 10 days of CUS, C57BL/6 mice exhibited a chronic-stress-associated corticosterone profile. Complete blood count (CBC) revealed mild normochromic, normocytic anemia, and reduced monocyte and lymphocyte count. Serum analysis demonstrated hypoferremia with unchanged total iron binding capacity and serum ferritin levels. These findings are consistent with clinical diagnostic parameters for anemia of chronic disease and indicate that CUS results in significant changes in blood and serum biochemical profile in C57BL/6 mice. These studies identify early changes in blood parameters in response to CUS and identify hematopoietic and biochemical alterations that are often associated with increased morbidity in patients experiencing chronic-stress-associated mental health disease.

Impacts of Psychological Stress on Osteoporosis: Clinical Implications and Treatment Interactions.

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.

Identification of circulating murine CD34+OCN+ cells.

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.

Non-linear hierarchy of the quorum sensing signalling pathway in bloodstream form African trypanosomes.

Trypanosoma brucei, the agents of African trypanosomiasis, undergo density-dependent differentiation in the mammalian bloodstream to prepare for transmission by tsetse flies. This involves the generation of cell-cycle arrested, quiescent, stumpy forms from proliferative slender forms. The signalling pathway responsible for the quorum sensing response has been catalogued using a genome-wide selective screen, providing a compendium of signalling protein kinases phosphatases, RNA binding proteins and hypothetical proteins. However, the ordering of these components is unknown. To piece together these components to provide a description of how stumpy formation arises we have used an extragenic suppression approach. This exploited a combinatorial gene knockout and overexpression strategy to assess whether the loss of developmental competence in null mutants of pathway components could be compensated by ectopic expression of other components. We have created null mutants for three genes in the stumpy induction factor signalling pathway (RBP7, YAK, MEKK1) and evaluated complementation by expression of RBP7, NEK17, PP1-6, or inducible gene silencing of the proposed differentiation inhibitor TbTOR4. This indicated that the signalling pathway is non-linear. Phosphoproteomic analysis focused on one pathway component, a putative MEKK, identified molecules with altered expression and phosphorylation profiles in MEKK1 null mutants, including another component in the pathway, NEK17. Our data provide a first molecular dissection of multiple components in a signal transduction cascade in trypanosomes.

Increased macrophage-derived SPARC precedes collagen deposition in myocardial fibrosis.

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.

hace1 Influences zebrafish cardiac development via ROS-dependent mechanisms.

BACKGROUND: In this study, we reveal a previously undescribed role of the HACE1 (HECT domain and Ankyrin repeat Containing E3 ubiquitin-protein ligase 1) tumor suppressor protein in normal vertebrate heart development using the zebrafish (Danio rerio) model. We examined the link between the cardiac phenotypes associated with hace1 loss of function to the expression of the Rho small family GTPase, rac1, which is a known target of HACE1 and promotes ROS production via its interaction with NADPH oxidase holoenzymes. RESULTS: We demonstrate that loss of hace1 in zebrafish via morpholino knockdown results in cardiac deformities, specifically a looping defect, where the heart is either tubular or "inverted". Whole-mount in situ hybridization of cardiac markers shows distinct abnormalities in ventricular morphology and atrioventricular valve formation in the hearts of these morphants, as well as increased expression of rac1. Importantly, this phenotype appears to be directly related to Nox enzyme-dependent ROS production, as both genetic inhibition by nox1 and nox2 morpholinos or pharmacologic rescue using ROS scavenging agents restores normal cardiac structure. CONCLUSIONS: Our study demonstrates that HACE1 is critical in the normal development and proper function of the vertebrate heart via a ROS-dependent mechanism. Developmental Dynamics 247:289-303, 2018. © 2017 Wiley Periodicals, Inc.

Elevated Wall Tension Initiates Interleukin-6 Expression and Abdominal Aortic Dilation.

BACKGROUND: 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). METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Role of a novel immune modulating DDR2-expressing population in silica-induced pulmonary fibrosis.

Micro-injuries associated with chronic inhaled particle exposures are linked with activation of the immune response and are thought to contribute to progression of fibrotic disease. In the pulmonary environment, we have previously demonstrated a heterogeneous population of circulating fibroblast precursors (CFPs), which are defined by expression of the pan-leukocyte marker CD45 and the collagen receptor, discoidin domain receptor-2 (DDR2). This population is derived from the hematopoietic stem cell, expresses collagen, and has a fibroblastic morphology in vitro. Herein, we demonstrate a novel subset of CFPs expressing immune markers CD11b, CD11c, and major histocompatibility complex II (MHC II). The CFP population was skewed toward this immune marker expressing subset in animals with silica-induced pulmonary fibrosis. Data indicate that this CFP subset upregulates co-stimulatory molecules and MHC II expression in response to silica-induced fibrosis in vivo. Functionally, this population was shown to promote T cell skewing away from a Th1 response and toward a pro-inflammatory profile. These studies represent the first direct flow cytometric and functional evaluation of the novel immune marker expressing CFP subset in an exposure-induced model of pulmonary fibrosis. Elucidating the role of this CFP subset may enhance our understanding of the complex immune balance critical to mediating exposures at the pulmonary-host interface and may be a valuable target for the treatment of exposure-induced pulmonary fibrosis.

Hematopoietic Origin of Murine Lung Fibroblasts.

Multiple origins, including the bone marrow, have been suggested to contribute to fibroblast populations in the lung. Using bone marrow reconstitution strategies, the present study tested the hypothesis that the bone marrow hematopoietic stem cell (HSC) gives rise to lung tissue fibroblasts in vivo. Data demonstrate that the nonadherent bone marrow fraction is enriched for CD45(+) HSC-derived cells and was able to reconstitute hematopoiesis in lethally irradiated animals. Analysis of peripheral blood and lung tissues from engrafted mice demonstrated the ability of this population to give rise to CD45(+)/Discoidin-Domain Receptor-2(+) (DDR2) circulating fibroblast precursors (CFPs) in blood and fibroblast populations in lung. An HSC origin for lung fibroblasts was confirmed using a novel clonal cell transplantation method in which the bone marrow is reconstituted by a clonal population derived from a single HSC. Together, these findings provide evidence for an HSC contribution to lung fibroblasts and demonstrate a circulating intermediate through the CD45(+)/DDR2(+) HSC-derived CFP.

Hematopoietic stem cell-derived cancer-associated fibroblasts are novel contributors to the pro-tumorigenic microenvironment.

Targeting the tumor microenvironment is critical toward improving the effectiveness of cancer therapeutics. Cancer-associated fibroblasts (CAFs) are one of the most abundant cell types of the tumor microenvironment, playing an important role in tumor progression. Multiple origins for CAFs have been proposed including resident fibroblasts, adipocytes, and bone marrow. Our laboratory previously identified a novel hematopoietic stem cell (HSC) origin for CAFs; however, the functional roles of HSC-derived CAFs (HSC-CAFs) in tumor progression have not yet been examined. To test the hypothesis that HSC-CAFs promote tumor progression through contribution to extracellular matrix (ECM) and paracrine production of pro-angiogenic factors, we developed a method to isolate HSC-CAFs. HSC-CAFs were profiled on the basis of their expression of hematopoietic and fibroblastic markers in two murine tumor models. Profiling revealed production of factors associated with ECM deposition and remodeling. Functional in vivo studies showed that co-injection of HSC-CAFs with tumor cells resulted in increased tumor growth rate and significantly larger tumors than tumor cells alone. Immunohistochemical studies revealed increased blood vessel density with co-injection, demonstrating a role for HSC-CAFs in tumor vascularization. Mechanistic in vitro studies indicated that HSC-CAFs play a role in producing vascular endothelial growth factor A and transforming growth factor-ß1 in endothelial tube formation and patterning. In vitro and in vivo findings suggest that HSC-CAFs are a critical component of the tumor microenvironment and suggest that targeting the novel HSC-CAF may be a promising therapeutic strategy.

Hematopoietic stem cell-derived adipocytes and fibroblasts in the tumor microenvironment.

The tumor microenvironment (TME) is complex and constantly evolving. This is due, in part, to the crosstalk between tumor cells and the multiple cell types that comprise the TME, which results in a heterogeneous population of tumor cells and TME cells. This review will focus on two stromal cell types, the cancer-associated adipocyte (CAA) and the cancer-associated fibroblast (CAF). In the clinic, the presence of CAAs and CAFs in the TME translates to poor prognosis in multiple tumor types. CAAs and CAFs have an activated phenotype and produce growth factors, inflammatory factors, cytokines, chemokines, extracellular matrix components, and proteases in an accelerated and aberrant fashion. Through this activated state, CAAs and CAFs remodel the TME, thereby driving all aspects of tumor progression, including tumor growth and survival, chemoresistance, tumor vascularization, tumor invasion, and tumor cell metastasis. Similarities in the tumor-promoting functions of CAAs and CAFs suggest that a multipronged therapeutic approach may be necessary to achieve maximal impact on disease. While CAAs and CAFs are thought to arise from tissues adjacent to the tumor, multiple alternative origins for CAAs and CAFs have recently been identified. Recent studies from our lab and others suggest that the hematopoietic stem cell, through the myeloid lineage, may serve as a progenitor for CAAs and CAFs. We hypothesize that the multiple origins of CAAs and CAFs may contribute to the heterogeneity seen in the TME. Thus, a better understanding of the origin of CAAs and CAFs, how this origin impacts their functions in the TME, and the temporal participation of uniquely originating TME cells may lead to novel or improved anti-tumor therapeutics.