Cytoplasmic retention of the DNA/RNA-binding protein FUS ameliorates organ fibrosis in mice.

Uncontrolled accumulation of extracellular matrix leads to tissue fibrosis and loss of organ function. We previously demonstrated in vitro that the DNA/RNA-binding protein fused in sarcoma (FUS) promotes fibrotic responses by translocating to the nucleus, where it initiates collagen gene transcription. However, it is still not known whether FUS is profibrotic in vivo and whether preventing its nuclear translocation might inhibit development of fibrosis following injury. We now demonstrate that levels of nuclear FUS are significantly increased in mouse models of kidney and liver fibrosis. To evaluate the direct role of FUS nuclear translocation in fibrosis, we used mice that carry a mutation in the FUS nuclear localization sequence (FUSR521G) and the cell-penetrating peptide CP-FUS-NLS that we previously showed inhibits FUS nuclear translocation in vitro. We provide evidence that FUSR521G mice or CP-FUS-NLS-treated mice showed reduced nuclear FUS and fibrosis following injury. Finally, differential gene expression analysis and immunohistochemistry of tissues from individuals with focal segmental glomerulosclerosis or nonalcoholic steatohepatitis revealed significant upregulation of FUS and/or collagen genes and FUS protein nuclear localization in diseased organs. These results demonstrate that injury-induced nuclear translocation of FUS contributes to fibrosis and highlight CP-FUS-NLS as a promising therapeutic option for organ fibrosis.

Human soluble prorenin receptor expressed in mouse renal collecting duct shows sex-specific effect on cardiorenal function.

Soluble prorenin receptor (sPRR), a component of the renin-angiotensin system (RAS), has been identified as a plasma biomarker for hypertension and cardiovascular diseases in humans. Despite studies showing that sPRR in the kidney is produced by tubular cells in the renal collecting duct (CD), its biological actions modulating cardiorenal function in physiological conditions remain unknown. Therefore, the objective of our study was to investigate whether CD-derived human sPRR (HsPRR) expression influences cardiorenal function and examine sex and circadian differences. Thus, we investigated the status of the intrarenal RAS, water and electrolyte balance, renal filtration capacity, and blood pressure (BP) regulation in CD-HsPRR and control (CTL) mice. CD-HsPRR mice were generated by breeding human sPRR-Myc-tag mice with Hoxb7/Cre mice. Renal sPRR expression increased in CD-HsPRR mice, but circulating sPRR and RAS levels were unchanged compared with CTL mice. Only female littermates expressing CD-HsPRR showed 1) increased 24-h BP, 2) an impaired BP response to an acute dose of losartan and attenuated angiotensin II (ANG II)-induced hypertension, 3) reduced angiotensin-converting enzyme activity and ANG II content in the renal cortex, and 4) decreased glomerular filtration rate, with no changes in natriuresis and kaliuresis despite upregulation of the ß-subunit of the epithelial Na+ channel in the renal cortex. These cardiorenal alterations were displayed only during the active phase of the day. Taken together, these data suggest that HsPRR could interact with ANG II type 1 receptors mediating sex-specific, ANG II-independent renal dysfunction and a prohypertensive phenotype in a sex-specific manner.NEW & NOTEWORTHY We successfully generated a humanized mouse model that expresses human sPRR in the collecting duct. Collecting duct-derived human sPRR did not change circulating sPRR and RAS levels but increased daytime BP in female mice while showing an attenuated angiotensin II-dependent pressor response. These findings may aid in elucidating the mechanisms by which women show uncontrolled BP in response to antihypertensive treatments targeting the RAS, improving approaches to reduce uncontrolled BP and chronic kidney disease incidences in women.

Tranexamic acid reduces inflammation, edema and burn wound conversion in a rodent model.

Burn wound conversion is the observed process where superficial partial thickness burns convert into deep partial or full thickness burn injuries. This conversion process often involves surgical excision to achieve timely wound healing. Unfortunately, the pathophysiology of this phenomenon is multifactorial and poorly understood. Thus, a therapeutic intervention that may prevent secondary progression and cell death in burn-injured tissue is desirable. Recent work by our group and others has established that tranexamic acid (TXA) has significant anti-inflammatory properties in addition to its well-known anti-fibrinolytic effects. This study investigates TXA as a novel therapeutic treatment to mitigate burn wound conversion and reduce systemic inflammation. Sprague-Dawley rats were subjected to a hot comb burn contact injury. A subset of animals underwent a similar comb burn with an adjacent 30%TBSA contact injury. The interspaces represent the ischemic zones simulating the zone of stasis. The treatment group received injections of TXA (100 mg/kg) immediately after injury and once daily until euthanasia. Animals were harvested for analyses at 6 h and 7 days after injury. Full-thickness biopsies from the ischemic zones and lung tissue were assessed with established histological techniques. Plasma was collected for measurement of damage associated molecular patterns (DAMPs), and liver samples were used to study inflammatory cytokines expression. Treatment with TXA was associated with reduced burn wound conversion and decreased burn-induced systemic inflammatory response syndrome (SIRS). Lung inflammation and capillary leak were also significantly reduced in TXA treated animals. Future research will elucidate the underlying anti-inflammatory properties of TXA responsible for these findings.

Human Soluble Prorenin Receptor Expressed in Adipose Tissue Improves Insulin Sensitivity and Endothelial Function in Obese Female Mice.

Increased circulating levels of the soluble prorenin receptor (sPRR), a component of the renin angiotensin system (RAS), plays a role in obesity, glucose, and insulin homeostasis. However, elevated plasma sPRR in diabetic patients has been shown correlated with hyperglycemia in women but not men. Hence, the current study sought to understand the contribution of human sPRR (HsPRR) produced in the adipose tissue (Adi) on adipogenesis, and glucose and insulin balance in obesity settings. Adi-HsPRR mice were generated by breeding human sPRR-Myc-tag transgenic mice with mice expressing Adiponectin/Cre. The mouse model was validated by detecting 28kDa myc-tagged HsPRR by western blotting. Adipose HsPRR expression did not change circulating sPRR in female mice fed a standard chow diet or high fat diet (HFD) but increased plasma sPRR in male Adi-HsPRR mice fed a HFD compared to HFD-fed controls. Yet, Adi-HsPRR improved insulin sensitivity, vascular relaxation and the vasodilator agent Ang 1-7 in obese female mice but not in the male counterparts. Moreover, Adi-HsPRR expression reduced the expression of the adipogenic genes SREBP1C and CD36 only in gonadal white adipose from obese female mice, signifying that adipose tissue-derived HsPRR exerts a sex-specific effect on insulin sensitivity and endothelial function which seems independent of circulating sPRR.

Activated CTHRC1 promotes glycolysis in endothelial cells: Implications for metabolism and angiogenesis.

CTHRC1 is transiently expressed by activated fibroblasts during tissue repair and in certain cancers, and CTHRC1 derived from osteocytes is detectable in circulation. Because its biological activity is poorly understood, we investigated whether the N terminus of CTHRC1 encodes a propeptide requiring cleavage to become activated. The effects of full-length versus cleaved recombinant CTHRC1 on endothelial cell metabolism and gene expression were examined in vitro. Respirometry was performed on Cthrc1 null and wildtype mice to obtain evidence for biological activity of CTHRC1 in vivo. Cleavage of the propeptide observed in vitro was attenuated in the presence of protease inhibitors, and cleaved CTHRC1 significantly promoted glycolysis whereas full-length CTHRC1 was less effective. The respiratory exchange ratio was significantly higher in wildtype mice compared to Cthrc1 null mice, supporting the findings of CTHRC1 promoting glycolysis in vivo. Key enzymes involved in glycolysis were significantly upregulated in endothelial cells in response to treatment with CTHRC1. In healthy human subjects, 58% of the cohort had detectable levels of circulating full-length CTHRC1, whereas all subjects with undetectable levels of full-length CTHRC1 (with one exception) had measurable levels of truncated CTHRC1 (88 pg/ml to >400 ng/ml). Our findings support a concept where CTHRC1 induction in activated fibroblasts at sites of ischemia such as tissue injury or cancer functions to increase glycolysis for ATP production under hypoxic conditions, thereby promoting cell survival and tissue repair. By promoting glycolysis under normoxic conditions, CTHRC1 may also be a contributor to the Warburg effect characteristically observed in many cancers.

Somatic variants of MAP3K3 are sufficient to cause cerebral and spinal cord cavernous malformations.

Cerebral cavernous malformations (CCMs) and spinal cord cavernous malformations (SCCMs) are common vascular abnormalities of the CNS that can lead to seizure, haemorrhage and other neurological deficits. Approximately 85% of patients present with sporadic (versus congenital) CCMs. Somatic mutations in MAP3K3 and PIK3CA were recently reported in patients with sporadic CCM, yet it remains unknown whether MAP3K3 mutation is sufficient to induce CCMs. Here we analysed whole-exome sequencing data for patients with CCM and found that ∼40% of them have a single, specific MAP3K3 mutation [c.1323C>G (p.Ile441Met)] but not any other known mutations in CCM-related genes. We developed a mouse model of CCM with MAP3K3I441M uniquely expressed in the endothelium of the CNS. We detected pathological phenotypes similar to those found in patients with MAP3K3I441M. The combination of in vivo imaging and genetic labelling revealed that CCMs were initiated with endothelial expansion followed by disruption of the blood-brain barrier. Experiments with our MAP3K3I441M mouse model demonstrated that CCM can be alleviated by treatment with rapamycin, the mTOR inhibitor. CCM pathogenesis has usually been attributed to acquisition of two or three distinct genetic mutations involving the genes CCM1/2/3 and/or PIK3CA. However, our results demonstrate that a single genetic hit is sufficient to cause CCMs.

Tgfß1-Cthrc1 Signaling Plays an Important Role in the Short-Term Reparative Response to Heart Valve Endothelial Injury.

OBJECTIVE: Aortic valve disease is a common worldwide health burden with limited treatment options. Studies have shown that the valve endothelium is critical for structure-function relationships, and disease is associated with its dysfunction, damage, or injury. Therefore, therapeutic targets to maintain a healthy endothelium or repair damaged endothelial cells could hold promise. In this current study, we utilize a surgical mouse model of heart valve endothelial cell injury to study the short-term response at molecular and cellular levels. The goal is to determine if the native heart valve exhibits a reparative response to injury and identify the mechanisms underlying this process. Approach and Results: Mild aortic valve endothelial injury and abrogated function was evoked by inserting a guidewire down the carotid artery of young (3 months) and aging (16-18 months) wild-type mice. Short-term cellular responses were examined at 6 hours, 48 hours, and 4 weeks following injury, whereas molecular profiles were determined after 48 hours by RNA-sequencing. Within 48 hours following endothelial injury, young wild-type mice restore endothelial barrier function in association with increased cell proliferation, and upregulation of transforming growth factor beta 1 (Tgfß1) and the glycoprotein, collagen triple helix repeat containing 1 (Cthrc1). Interestingly, this beneficial response to injury was not observed in aging mice with known underlying endothelial dysfunction. CONCLUSIONS: Data from this study suggests that the healthy valve has the capacity to respond to mild endothelial injury, which in short term has beneficial effects on restoring endothelial barrier function through acute activation of the Tgfß1-Cthrc1 signaling axis and cell proliferation.

Single-Cell RNA Sequencing Analysis Reveals a Crucial Role for CTHRC1 (Collagen Triple Helix Repeat Containing 1) Cardiac Fibroblasts After Myocardial Infarction.

BACKGROUND: Cardiac fibroblasts (CFs) have a central role in the ventricular remodeling process associated with different types of fibrosis. Recent studies have shown that fibroblasts do not respond homogeneously to heart injury. Because of the limited set of bona fide fibroblast markers, a proper characterization of fibroblast population heterogeneity in response to cardiac damage is lacking. The purpose of this study was to define CF heterogeneity during ventricular remodeling and the underlying mechanisms that regulate CF function. METHODS: Collagen1α1-GFP (green fluorescent protein)-positive CFs were characterized after myocardial infarction (MI) by single-cell and bulk RNA sequencing, assay for transposase-accessible chromatin sequencing, and functional assays. Swine and patient samples were studied using bulk RNA sequencing. RESULTS: We identified and characterized a unique CF subpopulation that emerges after MI in mice. These activated fibroblasts exhibit a clear profibrotic signature, express high levels of Cthrc1 (collagen triple helix repeat containing 1), and localize into the scar. Noncanonical transforming growth factor-ß signaling and different transcription factors including SOX9 are important regulators mediating their response to cardiac injury. Absence of CTHRC1 results in pronounced lethality attributable to ventricular rupture. A population of CFs with a similar transcriptome was identified in a swine model of MI and in heart tissue from patients with MI and dilated cardiomyopathy. CONCLUSIONS: We report CF heterogeneity and their dynamics during the course of MI and redefine the CFs that respond to cardiac injury and participate in myocardial remodeling. Our study identifies CTHRC1 as a novel regulator of the healing scar process and a target for future translational studies.

Activation of Skeletal Stem and Progenitor Cells for Bone Regeneration Is Driven by PDGFRß Signaling.

Bone repair and regeneration critically depend on the activation and recruitment of osteogenesis-competent skeletal stem and progenitor cells (SSPCs). Yet, the origin and triggering cues for SSPC propagation and migration remain largely elusive. Through bulk and single-cell transcriptome profiling of fetal osterix (Osx)-expressing cells, followed by lineage mapping, cell tracing, and conditional mouse mutagenesis, we here identified PDGF-PDGFRß signaling as critical functional mediator of SSPC expansion, migration, and angiotropism during bone repair. Our data show that cells marked by a history of Osx expression, including those arising in fetal or early postnatal periods, represent or include SSPCs capable of delivering all the necessary differentiated progeny to repair acute skeletal injuries later in life, provided that they express functional PDGFRß. Mechanistically, MMP-9 and VCAM-1 appear to be involved downstream of PDGF-PDGFRß. Our results reveal considerable cellular dynamism in the skeletal system and show that activation and recruitment of SSPCs for bone repair require functional PDGFRß signaling.

Reproducibility of CRISPR-Cas9 methods for generation of conditional mouse alleles: a multi-center evaluation.

BACKGROUND: CRISPR-Cas9 gene-editing technology has facilitated the generation of knockout mice, providing an alternative to cumbersome and time-consuming traditional embryonic stem cell-based methods. An earlier study reported up to 16% efficiency in generating conditional knockout (cKO or floxed) alleles by microinjection of 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides as donors (referred herein as "two-donor floxing" method). RESULTS: We re-evaluate the two-donor method from a consortium of 20 laboratories across the world. The dataset constitutes 56 genetic loci, 17,887 zygotes, and 1718 live-born mice, of which only 15 (0.87%) mice contain cKO alleles. We subject the dataset to statistical analyses and a machine learning algorithm, which reveals that none of the factors analyzed was predictive for the success of this method. We test some of the newer methods that use one-donor DNA on 18 loci for which the two-donor approach failed to produce cKO alleles. We find that the one-donor methods are 10- to 20-fold more efficient than the two-donor approach. CONCLUSION: We propose that the two-donor method lacks efficiency because it relies on two simultaneous recombination events in cis, an outcome that is dwarfed by pervasive accompanying undesired editing events. The methods that use one-donor DNA are fairly efficient as they rely on only one recombination event, and the probability of correct insertion of the donor cassette without unanticipated mutational events is much higher. Therefore, one-donor methods offer higher efficiencies for the routine generation of cKO animal models.

Bcl-2 Expression in Pericytes and Astrocytes Impacts Vascular Development and Homeostasis.

B-cell lymphoma 2 (Bcl-2) protein is the founding member of a group of proteins known to modulate apoptosis. Its discovery set the stage for identification of family members with either pro- or anti-apoptotic properties. Expression of Bcl-2 plays an important role during angiogenesis by influencing not only vascular cell survival, but also migration and adhesion. Although apoptosis and migration are postulated to have roles during vascular remodeling and regression, the contribution of Bcl-2 continues to emerge. We previously noted that the impaired retinal vascularization and an inability to undergo pathologic neovascularization observed in mice globally lacking Bcl-2 did not occur when mice lacked the expression of Bcl-2 only in endothelial cells. To further examine the effect of Bcl-2 expression during vascularization of the retina, we assessed its contribution in pericytes or astrocytes by generating mice with a conditional Bcl-2 allele (Bcl-2Flox/Flox) and Pdgfrb-cre (Bcl-2PC mice) or Gfap-cre (Bcl-2AC mice). Bcl-2PC and Bcl-2AC mice demonstrated increased retinal vascular cell apoptosis, reduced numbers of pericytes and endothelial cells and fewer arteries and veins in the retina. Bcl-2PC mice also demonstrated delayed advancement of the superficial retinal vascular layer and aberrant vascularization of the deep vascular plexus and central retina. Although pathologic neovascularization in oxygen-induced ischemic retinopathy (OIR) was not affected by lack of expression of Bcl-2 in either pericytes or astrocytes, laser-induced choroidal neovascularization (CNV) was significantly reduced in Bcl-2PC mice compared to littermate controls. Together these studies begin to reveal how cell autonomous modulation of apoptosis in vascular cells impacts development and homeostasis.

Targeted deletion of Cyp1b1 in pericytes results in attenuation of retinal neovascularization and trabecular meshwork dysgenesis.

Mutations in cytochrome P450 1B1 (CYP1B1) gene are reported in patients with primary congenital glaucoma. Cyp1b1-deficient (Cyp1b1-/-) mice show dysgenesis of the trabecular meshwork (TM) tissue and attenuation of retinal neovascularization during oxygen-induced ischemic retinopathy (OIR). Although retinal vascular cells, including endothelial cells (EC), pericytes (PC), astrocytes (AC), and TM endothelial cells express CYP1B1, the cell autonomous contribution of CYP1B1 to attenuation of retinal neovascularization and TM tissue dysgenesis remains unknown. Here we determined the impact lack of CYP1B1 expression in EC, PC or AC has on retinal neovascularization and TM tissue integrity. We generated Cyp1b1-transgenic mice with vascular cell-specific targeted Cre+-deletion in EC (Cyp1b1 EC), in PC (Cyp1b1 PC) and in AC (Cyp1b1 AC). Pathologic retinal neovascularization during OIR was evaluated by collagen IV staining of retinal wholemounts. Structural morphology of TM tissue was examined by transmission electron microscopy (TEM). The assessment of retinal neovascularization indicated a significant decrease in retinal neovascular tufts only in Cyp1b1 PC mice compared with control mice. TEM evaluation demonstrated Cyp1b1 PC mice also exhibited a defect in TM tissue morphology and integrity similar to that reported in Cyp1b1-/- mice. Thus, Cyp1b1 expression in PC plays a significant role in retinal neovascularization and the integrity of TM tissue.

Notch2 and Proteomic Signatures in Mouse Neointimal Lesion Formation.

OBJECTIVE: Vascular remodeling is associated with complex molecular changes, including increased Notch2, which promotes quiescence in human smooth muscle cells. We used unbiased protein profiling to understand molecular signatures related to neointimal lesion formation in the presence or absence of Notch2 and to test the hypothesis that loss of Notch2 would increase neointimal lesion formation because of a hyperproliferative injury response. APPROACH AND RESULTS: Murine carotid arteries isolated at 6 or 14 days after ligation injury were analyzed by mass spectrometry using a data-independent acquisition strategy in comparison to uninjured or sham injured arteries. We used a tamoxifen-inducible, cell-specific Cre recombinase strain to delete the Notch2 gene in smooth muscle cells. Vessel morphometric analysis and immunohistochemical staining were used to characterize lesion formation, assess vascular smooth muscle cell proliferation, and validate proteomic findings. Loss of Notch2 in smooth muscle cells leads to protein profile changes in the vessel wall during remodeling but does not alter overall lesion morphology or cell proliferation. Loss of smooth muscle Notch2 also decreases the expression of enhancer of rudimentary homolog, plectin, and annexin A2 in vascular remodeling. CONCLUSIONS: We identified unique protein signatures that represent temporal changes in the vessel wall during neointimal lesion formation in the presence and absence of Notch2. Overall lesion formation was not affected with loss of smooth muscle Notch2, suggesting compensatory pathways. We also validated the regulation of known injury- or Notch-related targets identified in other vascular contexts, providing additional insight into conserved pathways involved in vascular remodeling.

Bim expression in endothelial cells and pericytes is essential for regression of the fetal ocular vasculature.

Apoptosis plays a central role in developmental and pathological angiogenesis and vessel regression. Bim is a pro-apoptotic Bcl-2 family member that plays a prominent role in both developmental and pathological ocular vessel regression, and neovascularization. Endothelial cells (EC) and pericytes (PC) each play unique roles during vascular development, maintenance and regression. We recently showed that germline deletion of Bim results in persistent hyaloid vasculature, increased retinal vascular density and prevents retinal vessel regression in response to hyperoxia. To determine whether retinal vascular regression is attributable to Bim expression in EC or PC we generated mice carrying a conditional Bim allele (BimFlox/Flox) and VE-cadherin-cre (BimEC mice) or Pdgfrb-cre (BimPC mice). BimEC and BimPC mice demonstrated attenuated hyaloid vessel regression and postnatal retinal vascular remodeling. We also observed decreased retinal vascular apoptosis and proliferation. Unlike global Bim -/- mice, mice conditionally lacking Bim in EC or PC underwent hyperoxia-mediated vessel obliteration and subsequent retinal neovascularization during oxygen-induced ischemic retinopathy similar to control littermates. Thus, understanding the cell autonomous role Bim plays in the retinal vascular homeostasis will give us new insight into how to modulate pathological retinal neovascularization and vessel regression to preserve vision.

Inhibition of osteoclast differentiation and collagen antibody-induced arthritis by CTHRC1.

Collagen triple helix repeat-containing1 (Cthrc1) has previously been implicated in osteogenic differentiation and positive regulation of bone mass, however, the underlying mechanisms remain unclear. Here we characterized the bone phenotype of a novel Cthrc1 null mouse strain using bone histomorphometry, µCT analysis and functional readouts for bone strength. In male Cthrc1 null mice both trabecular bone as well as cortical bone formation was impaired, whereas in female Cthrc1 null mice only trabecular bone parameters were altered. Novel and highly specific monoclonal antibodies revealed that CTHRC1 is expressed by osteocytes and osteoblasts, but not osteoclasts. Furthermore, Cthrc1 null mice exhibited increased bone resorption with increased number of osteoclast and increased osteoclast activity together with enhanced expression of osteoclastogenic genes such as c-Fos, Rankl, Trap, and Nfatc1. Differentiation of bone marrow-derived monocytes isolated from Cthrc1 null mice differentiated into osteoclasts as effectively as those from wildtype mice. In the presence of CTHRC1 osteoclastogenic differentiation of bone marrow-derived monocytes was dramatically inhibited as was functional bone resorption by osteoclasts. This process was accompanied by downregulation of osteoclastogenic marker genes, indicating that extrinsically derived CTHRC1 is required for such activity. In vitro, CTHRC1 had no effect on osteogenic differentiation of bone marrow stromal cells, however, calvarial osteoblasts from Cthrc1 null mice exhibited reduced osteogenic differentiation compared to osteoblasts from wildtypes. In a collagen antibody-induced arthritis model Cthrc1 null mice suffered significantly more severe inflammation and joint destruction than wildtypes, suggesting that CTHRC1 expressed by the activated synoviocytes has anti-inflammatory effects. Mechanistically, we found that CTHRC1 inhibited NFκB activation by preventing IκBα degradation while also inhibiting ERK1/2 activation. Collectively our studies demonstrate that CTHRC1 secreted from osteocytes and osteoblasts functions as an inhibitor of osteoclast differentiation via inhibition of NFκB-dependent signaling. Furthermore, our data suggest that CTHRC1 has potent anti-inflammatory properties that limit arthritic joint destruction.

Visualization of Cancer and Cardiovascular Disease Co-Occurrence With Network Methods.

PURPOSE: Cancer and cardiovascular disease (CVD) are common causes of morbidity and mortality, and measurement and interpretation of their co-occurrence rate have important implications for public health and patient care. Here, we present the raw and adjusted co-occurrence rates of cancer and CVD in the overall population by using a visually intuitive network approach. METHODS: By using baseline survey and linked health outcome data from 490,842 individuals age 40 to 69 years from the UK Biobank, we recorded diagnoses between 1997 and 2014 of specific cancers and specific CVDs ascertained through hospital claims. We measured raw and adjusted rates of CVD for the following groups: individuals with Hodgkin or non-Hodgkin lymphoma, lung and trachea cancer, uterus cancer, colorectal cancer, prostate cancer, breast cancer, or no recorded diagnosed cancer during this time period. Analysis accounted for age, sex, and behavioral risk factors, without regard to the order of occurrence of cancer and CVD. RESULTS: A significantly increased rate of CVD was found in patients with multiple types of cancers, including Hodgkin and non-Hodgkin lymphoma and lung and trachea, uterus, colorectal, and breast cancer, compared with patients without cancer by using age and sex-adjusted models. Increased co-occurrence for many CVD categories remained after correction for behavioral risk factors. Construction of co-occurrence networks highlighted heart failure as a shared CVD diagnosis across multiple cancer types, including breast cancer, lung cancer, non-Hodgkin lymphoma, and colorectal cancer. Smoking, physical activity, and other lifestyle factors accounted for some but not all of the increased co-occurrence for many of the CVD diagnoses. CONCLUSION: Increased co-occurrence of several common CVD conditions is seen widely across multiple malignancies, and shared diagnoses, such as heart failure, were highlighted by using network methods.

Collagen triple helix repeat containing 1 is a new promigratory marker of arthritic pannus.

BACKGROUND: The formation of destructive hypercellular pannus is critical to joint damage in rheumatoid arthritis (RA). The collagen triple helix repeat containing 1 (CTHRC1) protein expressed by activated stromal cells of diverse origin has previously been implicated in tissue remodeling and carcinogenesis. We recently discovered that the synovial Cthrc1 mRNA directly correlates with arthritis severity in mice. This study characterizes the role of CTHRC1 in arthritic pannus formation. METHODS: Synovial joints of mice with collagen antibody-induced arthritis (CAIA) and human RA-fibroblast-like synoviocytes (FLS) were immunostained for CTHRC1, FLS and macrophage-specific markers. CTHRC1 levels in plasma from patients with RA were measured using sandwich ELISA. The migratory response of fibroblasts was studied with a transwell migration assay and time-lapse microscopy. Velocity and directness of cell migration was analyzed by recording the trajectories of cells treated with rhCTHRC1. RESULTS: Immunohistochemical analysis of normal and inflamed synovium revealed highly inducible expression of CTHRC1 in arthritis (10.9-fold). At the tissue level, CTHRC1-expressing cells occupied the same niche as large fibroblast-like cells positive for α-smooth muscle actin (α-SMA) and cadherin 11 (CDH11). CTHRC1 was produced by activated FLS predominantly located at the synovial intimal lining and at the bone-pannus interface. Cultured RA-FLS expressed CDH11, α-SMA, and CTHRC1. Upon treatment with exogenous rhCTHRC1, embryonic fibroblasts and RA-FLS significantly increased migration velocity, directness, and cell length along the front-tail axis (1.4-fold, p < 0.01). CONCLUSION: CTHRC1 was established as a novel marker of activated synoviocytes in murine experimental arthritis and RA. The pro-migratory effect of CTHRC1 on synoviocytes is considered one of the mechanisms promoting hypercellularity of the arthritic pannus.

Lipid Profiling of In Vitro Cell Models of Adipogenic Differentiation: Relationships With Mouse Adipose Tissues.

Our objective was to characterize lipid profiles in cell models of adipocyte differentiation in comparison to mouse adipose tissues in vivo. A novel lipid extraction strategy was combined with global lipid profiling using direct infusion and sequential precursor ion fragmentation, termed MS/MS(ALL) . Perirenal and inguinal white adipose tissue and interscapular brown adipose tissues from adult C57BL/6J mice were analyzed. 3T3-L1 preadipocytes, ear mesenchymal progenitor cells, and brown adipose-derived BAT-C1 cells were also characterized. Over 3000 unique lipid species were quantified. Principal component analysis showed that perirenal versus inguinal white adipose tissues varied in lipid composition of triacyl- and diacylglycerols, sphingomyelins, glycerophospholipids and, notably, cardiolipin CL 72:3. In contrast, hexosylceramides and sphingomyelins distinguished brown from white adipose. Adipocyte differentiation models showed broad differences in lipid composition among themselves, upon adipogenic differentiation, and with adipose tissues. Palmitoyl triacylglycerides predominate in 3T3-L1 differentiation models, whereas cardiolipin CL 72:1 and SM 45:4 were abundant in brown adipose-derived cell differentiation models, respectively. MS/MS(ALL) data suggest new lipid biomarkers for tissue-specific lipid contributions to adipogenesis, thus providing a foundation for using in vitro models of adipogenesis to reflect potential changes in adipose tissues in vivo. J. Cell. Biochem. 117: 2182-2193, 2016. © 2016 Wiley Periodicals, Inc.

Cthrc1 controls adipose tissue formation, body composition, and physical activity.

OBJECTIVE: This study investigated the effects of loss of Cthrc1 on adipogenesis, body composition, metabolism, physical activity, and muscle physiology. METHODS: Complete metabolic and activity monitoring as well as grip strength measurements and muscle myography was performed in Cthrc1 null and wildtype mice. RESULTS: Compared to wildtypes, Cthrc1 null mice had similar body weights but significantly reduced energy expenditure, decreased lean mass, and increased fat mass, especially visceral fat. In vitro studies demonstrated that Cthrc1 inhibited adipocyte differentiation as well as PPAR and CREB reporter activity, while preadipocytes isolated from Cthrc1 null mice exhibited enhanced adipogenic differentiation. Voluntary physical activity in Cthrc1 null mice as assessed by wheel running was reduced to approximately half the distance covered by wildtypes. Reduced grip strength was observed in Cthrc1 null mice at the age of 15 weeks or older with reduced performance and mass of hyphenate muscle. In the brain, Cthrc1 expression was most prominent in neurons of thalamic and hypothalamic nuclei with evidence for secretion into the circulation in the median eminence. CONCLUSIONS: Our data indicate that Cthrc1 regulates body composition through inhibition of adipogenesis. In addition, central Cthrc1 may be a mediator of muscle function and physical activity.