Macrophages preserve endothelial cell specialization in the adrenal gland to modulate aldosterone secretion and blood pressure.

Macrophages play crucial roles in organ-specific functions and homeostasis. In the adrenal gland, macrophages closely associate with sinusoidal capillaries in the aldosterone-producing zona glomerulosa. We demonstrate that macrophages preserve capillary specialization and modulate aldosterone secretion. Using macrophage-specific deletion of VEGF-A, single-cell transcriptomics, and functional phenotyping, we found that the loss of VEGF-A depletes PLVAP+ fenestrated endothelial cells in the zona glomerulosa, leading to increased basement membrane collagen IV deposition and subendothelial fibrosis. This results in increased aldosterone secretion, called "haptosecretagogue" signaling. Human aldosterone-producing adenomas also show capillary rarefaction and basement membrane thickening. Mice with myeloid cell-specific VEGF-A deletion exhibit elevated serum aldosterone, hypokalemia, and hypertension, mimicking primary aldosteronism. These findings underscore macrophage-to-endothelial cell signaling as essential for endothelial cell specialization, adrenal gland function, and blood pressure regulation, with broader implications for other endocrine organs.

The regulatory effect of hyaluronan on human mesenchymal stem cells' fate modulates their interaction with cancer cells in vitro.

Metastatic spread of cancer cells into a pre-metastatic niche is highly dependent on a supporting microenvironment. Human bone marrow-derived mesenchymal stem cells (bmMSCs) contribute to the tumor microenvironment and promote cancer metastasis by inducing epithelial-to-mesenchymal transition and immune evasion. The underlying mechanisms, however, are incompletely understood. The glycosaminoglycan hyaluronan (HA) is a central component of the extracellular matrix and has been shown to harbor pro-metastatic properties. In this study we investigated the highly disseminating breast cancer and glioblastoma multiforme cell lines MDA-MB-321 and U87-MG which strongly differ in their metastatic potential to evaluate the impact of HA on tumor promoting features of bmMSC and their interaction with tumor cells. We show that adipogenic differentiation of bmMSC is regulated by the HA-matrix. This study reveals that MDA-MB-231 cells inhibit this process by the induction of HA-synthesis in bmMSCs and thus preserve the pro-tumorigenic properties of bmMSC. Furthermore, we show that adhesion of MDA-MB-231 cells to bmMSC is facilitated by the tumor cell-induced HA-rich matrix and is mediated by the HA-receptor LAYN. We postulate that invasive breast cancer cells modulate the HA-matrix of bmMSC to adapt the pre-metastatic niche. Thus, the HA-matrix provides a potential novel therapeutic target to prevent cancer metastasis.

Single-cell transcriptomics defines heterogeneity of epicardial cells and fibroblasts within the infarcted murine heart.

In the adult heart, the epicardium becomes activated after injury, contributing to cardiac healing by secretion of paracrine factors. Here, we analyzed by single-cell RNA sequencing combined with RNA in situ hybridization and lineage tracing of Wilms tumor protein 1-positive (WT1+) cells, the cellular composition, location, and hierarchy of epicardial stromal cells (EpiSC) in comparison to activated myocardial fibroblasts/stromal cells in infarcted mouse hearts. We identified 11 transcriptionally distinct EpiSC populations, which can be classified into three groups, each containing a cluster of proliferating cells. Two groups expressed cardiac specification markers and sarcomeric proteins suggestive of cardiomyogenic potential. Transcripts of hypoxia-inducible factor (HIF)-1α and HIF-responsive genes were enriched in EpiSC consistent with an epicardial hypoxic niche. Expression of paracrine factors was not limited to WT1+ cells but was a general feature of activated cardiac stromal cells. Our findings provide the cellular framework by which myocardial ischemia may trigger in EpiSC the formation of cardioprotective/regenerative responses.

4-Methylumbelliferone Attenuates Macrophage Invasion and Myocardial Remodeling in Pressure-Overloaded Mouse Hearts.

[Figure: see text].

Helix-Loop-Helix Factor Id3 (Inhibitor of Differentiation 3): A Novel Regulator of Hyaluronan-Mediated Adipose Tissue Inflammation.

OBJECTIVE: The aim of this study was to unravel mechanisms whereby deficiency of the transcription factor Id3 (inhibitor of differentiation 3) leads to metabolic dysfunction in visceral obesity. We investigated the impact of loss of Id3 on hyaluronic acid (HA) production by the 3 HAS isoenzymes (HA synthases; -1, -2, and -3) and on obesity-induced adipose tissue (AT) accumulation of proinflammatory B cells. Approach and Results: Male Id3-/- mice and respective wild-type littermate controls were fed a 60% high-fat diet for 4 weeks. An increase in inflammatory B2 cells was detected in Id3-/- epididymal AT. HA accumulated in epididymal AT of high-fat diet-fed Id3-/- mice and circulating levels of HA were elevated. Has2 mRNA expression was increased in epididymal AT of Id3-/- mice. Luciferase promoter assays showed that Id3 suppressed Has2 promoter activity, while loss of Id3 stimulated Has2 promoter activity. Functionally, HA strongly promoted B2 cell adhesion in the AT and on cultured vascular smooth muscle cells of Id3-/- mice, an effect sensitive to hyaluronidase. CONCLUSIONS: Our data demonstrate that loss of Id3 increases Has2 expression in the epididymal AT, thereby promoting HA accumulation. In turn, elevated HA content promotes HA-dependent binding of B2 cells and an increase in the B2 cells in the AT, which contributes to AT inflammation.

Intra-Patient Heterogeneity of Circulating Tumor Cells and Circulating Tumor DNA in Blood of Melanoma Patients.

Despite remarkable progress in melanoma therapy, the exceptional heterogeneity of the disease has prevented the development of reliable companion biomarkers for the prediction or monitoring of therapy responses. Here, we show that difficulties in detecting blood-based markers, like circulating tumor cells (CTC), might arise from the translation of the mutational heterogeneity of melanoma cells towards their surface marker expression. We provide a unique method, which enables the molecular characterization of clinically relevant CTC subsets, as well as circulating tumor DNA (ctDNA), from a single blood sample. The study demonstrates the benefit of a combined analysis of ctDNA and CTC counts in melanoma patients, revealing that CTC subsets and ctDNA provide synergistic real-time information on the mutational status, RNA and protein expression of melanoma cells in individual patients, in relation to clinical outcome.

Inhibition of the hyaluronan matrix enhances metabolic anticancer therapy by dichloroacetate in vitro and in vivo.

BACKGROUND AND PURPOSE: Aerobic glycolysis is a unique feature of tumour cells that entails several advantages for cancer progression such as resistance to apoptosis. The low MW compound, dichloroacetate, is a pyruvate dehydrogenase kinase inhibitor, which restores oxidative phosphorylation and induces apoptosis in a variety of cancer entities. However, its therapeutic effectiveness is limited by resistance mechanisms. This study aimed to examine the role of the anti-apoptotic hyaluronan (HA) matrix in this context and to identify a potential add-on treatment option to overcome this limitation. EXPERIMENTAL APPROACH: The metabolic connection between dichloroacetate treatment and HA matrix augmentation was analysed in vitro by quantitative PCR and affinity cytochemistry. Metabolic pathways were analysed using Seahorse, HPLC, fluorophore-assisted carbohydrate electrophoresis, colourimetry, immunoblots, and immunochemistry. The effects of combining dichloroacetate with the HA synthesis inhibitor 4-methylumbelliferone was evaluated in 2D and 3D cell cultures and in a nude mouse tumour xenograft regression model by immunoblot, immunochemistry, and FACS analysis. KEY RESULTS: Mitochondrial reactivation induced by dichloroacetate metabolically activated HA synthesis by augmenting precursors as well as O-GlcNAcylation. This process was blocked by 4-methylumbelliferone, resulting in enhanced anti-tumour efficacy in 2D and 3D cell culture and in a nude mouse tumour xenograft regression model. CONCLUSIONS AND IMPLICATIONS: The HA rich tumour micro-environment represents a metabolic factor contributing to chemotherapy resistance. HA synthesis inhibition exhibited pronounced synergistic actions with dichloroacetate treatment on oesophageal tumour cell proliferation and survival in vitro and in vivo suggesting the combination of these two strategies is an effective anticancer therapy.

Decreased M1 macrophage polarization in dabigatran-treated Ldlr-deficient mice: Implications for atherosclerosis and adipose tissue inflammation.

BACKGROUND AND AIMS: The non-vitamin K oral anticoagulant dabigatran etexilate (dabigatran) is increasingly prescribed to patients with non-valvular atrial fibrillation and venous thromboembolism. Adipose tissue (AT) inflammation during obesity plays a crucial role in the development of insulin resistance, type II diabetes and atherogenesis. The aim of the present study was to investigate the effects of thrombin inhibition by dabigatran in a combined model of diet-induced obesity and atherosclerosis. METHODS: Female Low density lipoprotein receptor knockout (Lldr-/-) mice were fed a high-fat diet containing 5 mg/g dabigatran or matching control for 20 weeks. RESULTS: Dabigatran-treated animals showed increased adipocyte hypertrophy, but reduced numbers of pro-inflammatory M1-polarized macrophages in the adipose tissue. Abundance of pro-inflammatory M1 macrophages was also decreased in the aortic wall of dabigatran-fed mice. Multiple circulating cytokines were reduced, indicating an effect in systemically relevant secretory compartments such as the AT. CONCLUSIONS: Dabigatran treatment reduces pro-inflammatory M1 macrophages in atherosclerotic lesions, thereby contributing to plaque stabilizing and atheroprotective effects of the thrombin inhibitor. This finding is not restricted to the vascular wall but is also present in AT where dabigatran treatment reduced the release of pro-inflammatory cytokines and accumulation of M1 macrophages.

Impact of hyperinsulinemia and hyperglycemia on valvular interstitial cells - A link between aortic heart valve degeneration and type 2 diabetes.

Type 2 diabetes is a known risk factor for cardiovascular diseases and is associated with an increased risk to develop aortic heart valve degeneration. Nevertheless, molecular mechanisms leading to the pathogenesis of valve degeneration in the context of diabetes are still not clear. Hence, we hypothesized that classical key factors of type 2 diabetes, hyperinsulinemia and hyperglycemia, may affect signaling, metabolism and degenerative processes of valvular interstitial cells (VIC), the main cell type of heart valves. Therefore, VIC were derived from sheep and were treated with hyperinsulinemia, hyperglycemia and the combination of both. The presence of insulin receptors was shown and insulin led to increased proliferation of the cells, whereas hyperglycemia alone showed no effect. Disturbed insulin response was shown by impaired insulin signaling, i.e. by decreased phosphorylation of Akt/GSK-3α/ß pathway. Analysis of glucose transporter expression revealed absence of glucose transporter 4 with glucose transporter 1 being the predominantly expressed transporter. Glucose uptake was not impaired by disturbed insulin response, but was increased by hyperinsulinemia and was decreased by hyperglycemia. Analyses of glycolysis and mitochondrial respiration revealed that VIC react with increased activity to hyperinsulinemia or hyperglycemia, but not to the combination of both. VIC do not show morphological changes and do not acquire an osteogenic phenotype by hyperinsulinemia or hyperglycemia. However, the treatment leads to increased collagen type 1 and decreased α-smooth muscle actin expression. This work implicates a possible role of diabetes in early phases of the degeneration of aortic heart valves.

Cardiac Hyaluronan Synthesis Is Critically Involved in the Cardiac Macrophage Response and Promotes Healing After Ischemia Reperfusion Injury.

RATIONALE: Immediate changes in the ECM (extracellular matrix) microenvironment occur after myocardial ischemia and reperfusion (I/R) injury. OBJECTIVE: Aim of this study was to unravel the role of the early hyaluronan (HA)-rich ECM after I/R. METHODS AND RESULTS: Genetic deletion of Has2 and Has1 was used in a murine model of cardiac I/R. Chemical exchange saturation transfer imaging was adapted to image cardiac ECM post-I/R. Of note, the cardiac chemical exchange saturation transfer signal was severely suppressed by Has2 deletion and pharmacological inhibition of HA synthesis 24 hours after I/R. Has2 KO ( Has2 deficient) mice showed impaired hemodynamic function suggesting a protective role for endogenous HA synthesis. In contrast to Has2 deficiency, Has1-deficient mice developed no specific phenotype compared with control post-I/R. Importantly, in Has2 KO mice, cardiac macrophages were diminished after I/R as detected by 19F MRI (magnetic resonance imaging) of perfluorcarbon-labeled immune cells, Mac-2/Galectin-3 immunostaining, and FACS (fluorescence-activated cell sorting) analysis (CD45+CD11b+Ly6G-CD64+F4/80+cells). In contrast to macrophages, cardiac Ly6Chigh and Ly6Clow monocytes were unaffected post-I/R compared with control mice. Mechanistically, inhibition of HA synthesis led to increased macrophage apoptosis in vivo and in vitro. In addition, α-SMA (α-smooth muscle actin)-positive cells were reduced in the infarcted myocardium and in the border zone. In vitro, the myofibroblast response as measured by Acta2 mRNA expression was reduced by inhibition of HA synthesis and of CD44 signaling. Furthermore, Has2 KO fibroblasts were less able to contract collagen gels in vitro. The effects of HA/CD44 on fibroblasts and macrophages post-I/R might also affect intercellular cross talk because cardiac fibroblasts were activated by monocyte/macrophages and, in turn, protected macrophages from apoptosis. CONCLUSIONS: Increased HA synthesis contributes to postinfarct healing by supporting macrophage survival and by promoting the myofibroblast response. Additionally, imaging of cardiac HA by chemical exchange saturation transfer post-I/R might have translational value.

Degenerative aortic valve disease and diabetes: Implications for a link between proteoglycans and diabetic disorders in the aortic valve.

Degenerative aortic valve disease in combination with diabetes is an increasing burden worldwide. There is growing evidence that particularly small leucine-rich proteoglycans are involved in the development of degenerative aortic valve disease. Nevertheless, the role of these molecules in this disease in the course of diabetes has not been elucidated in detail and previous studies remain controversial. Therefore, the aim of this study is to broaden the knowledge about small leucine-rich proteoglycans in degenerative aortic valve disease and the influence of diabetes and hyperglycaemia on aortic valves and valvular interstitial cells is examined. Analyses were performed using reverse-transcription polymerase chain reaction, Western blot, enzyme-linked immunosorbent assay, (immuno)histology and colorimetric assays. We could show that biglycan, but not decorin and lumican, is upregulated in degenerated human aortic valve cusps. Subgroup analysis reveals that upregulation of biglycan is stage-dependent. In vivo, loss of biglycan leads to stage-dependent calcification and also to migratory effects on interstitial cells within the extracellular matrix. In late stages of degenerative aortic valve disease, diabetes increases the expression of biglycan in aortic valves. In vitro, the combinations of hyperglycaemic with pro-degenerative conditions lead to an upregulation of biglycan. In conclusion, biglycan represents a potential link between degenerative aortic valve disease and diabetes.

Inhibition of Radiation-Induced Ccl2 Signaling Protects Lungs from Vascular Dysfunction and Endothelial Cell Loss.

Aims: Radiation-induced normal tissue toxicity often precludes the application of curative radiation doses. Here we investigated the therapeutic potential of chemokine C-C motif ligand 2 (Ccl2) signaling inhibition to protect normal lung tissue from radiotherapy (RT)-induced injury. Results: RT-induced vascular dysfunction and associated adverse effects can be efficiently antagonized by inhibition of Ccl2 signaling using either the selective Ccl2 inhibitor bindarit (BIN) or mice deficient for the main Ccl2 receptor CCR2 (KO). BIN-treatment efficiently counteracted the RT-induced expression of Ccl2, normalized endothelial cell (EC) morphology and vascular function, and limited lung inflammation and metastasis early after irradiation (acute effects). A similar protection of the vascular compartment was detected by loss of Ccl2 signaling in lungs of CCR2-KO mice. Long-term Ccl2 signaling inhibition also significantly limited EC loss and accompanied fibrosis progression as adverse late effect. With respect to the human situation, we further confirmed that Ccl2 secreted by RT-induced senescent epithelial cells resulted in the activation of normally quiescent but DNA-damaged EC finally leading to EC loss in ex vivo cultured human normal lung tissue. Innovation: Abrogation of certain aspects of the secretome of irradiated resident lung cells, in particular signaling inhibition of the senescence-associated secretory phenotype-factor Ccl2 secreted predominantly by RT-induced senescent epithelial cells, resulted in protection of the endothelial compartment. Conclusions: Radioprotection of the normal tissue via Ccl2 signaling inhibition without simultaneous protection or preferable radiosensitization of tumor tissue might improve local tumor control and survival, because higher doses of radiation could be used.

Enzymatic Activity Is Not Required for Phospholipase D Mediated TNF-α Regulation and Myocardial Healing.

Phospholipase D1 is a regulator of tumor necrosis factor-α expression and release upon LPS-induced sepsis and following myocardial infarction (MI). Lack of PLD1 leads to a reduced TNF-α mediated inflammatory response and to enhanced infarct size with declined cardiac function 21 days after ischemia reperfusion (I/R) injury. Deficiency of both PLD isoforms PLD1 and PLD2 as well as pharmacological inhibition of the enzymatic activity of PLD with the PLD inhibitor FIPI protected mice from arterial thrombosis and ischemic brain infarction. Here we treated mice with the PLD inhibitor FIPI to analyze if pharmacological inhibition of PLD after myocardial ischemia protects mice from cardiac damage. Inhibition of PLD with FIPI leads to reduced migration of inflammatory cells into the infarct border zone 24 h after experimental MI in mice, providing first evidence for immune cell migration to be dependent on the enzymatic activity of PLD. In contrast to PLD1 deficient mice, TNF-α plasma level was not altered after FIPI treatment of mice. Consequently, infarct size and left ventricular (LV) function were comparable between FIPI-treated and control mice 21 days post MI. Moreover, cell survival 24 h post I/R was not altered upon FIPI treatment. Our results indicate that the enzymatic activity of PLD is not responsible for PLD mediated TNF-α signaling and myocardial healing after I/R injury in mice. Furthermore, reduced TNF-α plasma levels in PLD1 deficient mice might be responsible for increased infarct size and impaired cardiac function 21 days post MI.

CDKN1B/p27 is localized in mitochondria and improves respiration-dependent processes in the cardiovascular system-New mode of action for caffeine.

We show that the cyclin-dependent kinase inhibitor 1B (CDKN1B)/p27, previously known as a cell cycle inhibitor, is also localized within mitochondria. The migratory capacity of endothelial cells, which need intact mitochondria, is completely dependent on mitochondrial p27. Mitochondrial p27 improves mitochondrial membrane potential, increases adenosine triphosphate (ATP) content, and is required for the promigratory effect of caffeine. Domain mapping of p27 revealed that the N-terminus and C-terminus are required for those improvements. Further analysis of those regions revealed that the translocation of p27 into the mitochondria and its promigratory activity depend on serine 10 and threonine 187. In addition, mitochondrial p27 protects cardiomyocytes against apoptosis. Moreover, mitochondrial p27 is necessary and sufficient for cardiac myofibroblast differentiation. In addition, p27 deficiency and aging decrease respiration in heart mitochondria. Caffeine does not increase respiration in p27-deficient animals, whereas aged mice display improvement after 10 days of caffeine in drinking water. Moreover, caffeine induces transcriptome changes in a p27-dependent manner, affecting mostly genes relevant for mitochondrial processes. Caffeine also reduces infarct size after myocardial infarction in prediabetic mice and increases mitochondrial p27. Our data characterize mitochondrial p27 as a common denominator that improves mitochondria-dependent processes and define an increase in mitochondrial p27 as a new mode of action of caffeine.

Targeting sphingosine-1-phosphate lyase as an anabolic therapy for bone loss.

Sphingosine-1-phosphate (S1P) signaling influences bone metabolism, but its therapeutic potential in bone disorders has remained unexplored. We show that raising S1P levels in adult mice through conditionally deleting or pharmacologically inhibiting S1P lyase, the sole enzyme responsible for irreversibly degrading S1P, markedly increased bone formation, mass and strength and substantially decreased white adipose tissue. S1P signaling through S1P2 potently stimulated osteoblastogenesis at the expense of adipogenesis by inversely regulating osterix and PPAR-γ, and it simultaneously inhibited osteoclastogenesis by inducing osteoprotegerin through newly discovered p38-GSK3ß-ß-catenin and WNT5A-LRP5 pathways. Accordingly, S1P2-deficient mice were osteopenic and obese. In ovariectomy-induced osteopenia, S1P lyase inhibition was as effective as intermittent parathyroid hormone (iPTH) treatment in increasing bone mass and was superior to iPTH in enhancing bone strength. Furthermore, lyase inhibition in mice successfully corrected severe genetic osteoporosis caused by osteoprotegerin deficiency. Human data from 4,091 participants of the SHIP-Trend population-based study revealed a positive association between serum levels of S1P and bone formation markers, but not resorption markers. Furthermore, serum S1P levels were positively associated with serum calcium , negatively with PTH , and curvilinearly with body mass index. Bone stiffness, as determined through quantitative ultrasound, was inversely related to levels of both S1P and the bone formation marker PINP, suggesting that S1P stimulates osteoanabolic activity to counteract decreasing bone quality. S1P-based drugs should be considered as a promising therapeutic avenue for the treatment of osteoporotic diseases.

Hyaluronan synthase 3 promotes plaque inflammation and atheroprogression.

OBJECTIVE: Hyaluronan (HA) is a prominent component of the provisional extracellular matrix (ECM) present in the neointima of atherosclerotic plaques. Here the role of HA synthase 3 (HAS3) in atheroprogression was studied. APPROACH AND RESULTS: It is demonstrated here that HAS isoenzymes 1, -2 and -3 are expressed in human atherosclerotic plaques of the carotid artery. In Apolipoprotein E (Apoe)-deficient mice Has3 expression is increased early during lesion formation when macrophages enter atherosclerotic plaques. Importantly, HAS3 expression in vascular smooth muscle cells (VSMC) was found to be regulated by interleukin 1 ß (IL-1ß) in an NFkB dependent manner and blocking antibodies to IL-1ß abrogate Has3 expression in VSMC by activated macrophages. Has3/Apoe double deficient mice developed less atherosclerosis characterized by decreased Th1-cell responses, decreased IL-12 release, and decreased macrophage-driven inflammation. CONCLUSIONS: Inhibition of HAS3-dependent synthesis of HA dampens systemic Th1 cell polarization and reduces plaque inflammation. These data suggest that HAS3 might be a promising therapeutic target in atherosclerosis. Moreover, because HAS3 is regulated by IL-1ß, our results suggest that therapeutic anti-IL-1ß antibodies, recently tested in human clinical trials (CANTOS), may exert their beneficial effects on inflammation in post-myocardial infarction patients in part via effects on HAS3. TOC categorybasic study TOC subcategoryarteriosclerosis.

Hyperglycaemia and aberrated insulin signalling stimulate tumour progression via induction of the extracellular matrix component hyaluronan.

Epidemiological studies have detected a higher incidence of various tumour entities in diabetic patients. However, the underlying mechanisms remain insufficiently understood. Glucose-derived pericellular and extracellular hyaluronan (HA) promotes tumour progression and development. In our study, we tested the hypothesis that a diabetic metabolic state, characterised by hyperglycaemia and concomitant aberrant insulin signalling, stimulates tumour progression via the induction of HA synthesis. In a streptozotocin-induced diabetic nude mouse tumour xenograft model, hyperglycaemia and lack of insulin caused an increased formation of tumour-associated HA-matrix, which in turn accelerated tumour progression and neoangiogenesis. This process was effectively attenuated by treatment with 4-methylumbelliferone, a pharmacological inhibitor of HA-synthesis. To define the mechanisms behind these in vivo observations, we investigated the impact of hyperglycaemia and insulin on the glucose metabolism in oesophageal squamous cell cancer cells (ESCC). Hyperglycaemia induced HA synthesis while insulin diminished HA production by directing glucose metabolites to glycolysis. Vice versa, inhibition of glycolysis, either by knockdown of the glycolytic key enzyme phosphofructokinase or by an experimental abrogation of insulin signalling (knockdown of the insulin receptor and long-term treatment with insulin) augmented HA synthesis. Consequently, these processes induced invasion, anchorage-independent growth and adhesion of ESCC to endothelial cells in vitro. Thus, the cellular shift in glucose usage from catabolism of glucose to anabolism of HA driven by hyperglycaemia and insulin resistance may represent an important link between diabetes and cancer progression. Hence, therapeutical inhibition of HA synthesis may represent a promising approach for tumour treatment in diabetic patients.

Loss of CD73 prevents accumulation of alternatively activated macrophages and the formation of prefibrotic macrophage clusters in irradiated lungs.

While radiotherapy is a mainstay for cancer therapy, pneumonitis and fibrosis constitute dose-limiting side effects of thorax and whole body irradiation. So far, the contribution of immune cells to disease progression is largely unknown. Here we studied the role of ecto-5'-nucelotidase (CD73)/adenosine-induced changes in the myeloid compartment in radiation-induced lung fibrosis. C57BL/6 wild-type or CD73-/- mice received a single dose of whole thorax irradiation (WTI, 15 Gy). Myeloid cells were characterized in flow cytometric, histologic, and immunohistochemical analyses as well as RNA analyses. WTI induced a pronounced reduction of alveolar macrophages in both strains that recovered within 6 wk. Fibrosis development in wild-type mice was associated with a time-dependent deposition of hyaluronic acid (HA) and increased expression of markers for alternative activation on alveolar macrophages. These include the antiinflammatory macrophage mannose receptor and arginase-1. Further, macrophages accumulated in organized clusters and expressed profibrotic mediators at ≥25 wk after irradiation (fibrotic phase). Irradiated CD73-/- mice showed an altered regulation of components of the HA system and no clusters of alternatively activated macrophages. We speculate that accumulation of alternatively activated macrophages in organized clusters represents the origins of fibrotic foci after WTI and is promoted by a cross-talk between HA, CD73/adenosine signaling, and other profibrotic mediators.-De Leve, S., Wirsdörfer, F., Cappuccini, F., Schütze, A., Meyer, A. V., Röck, K., Thompson, L. F., Fischer, J. W., Stuschke, M., Jendrossek, V. Loss of CD73 prevents accumulation of alternatively activated macrophages and the formation of prefibrotic macrophage clusters in irradiated lungs.

Mesenchymal Stem Cell Therapy Protects Lungs from Radiation-Induced Endothelial Cell Loss by Restoring Superoxide Dismutase 1 Expression.

AIMS: Radiation-induced normal tissue toxicity is closely linked to endothelial cell (EC) damage and dysfunction (acute effects). However, the underlying mechanisms of radiation-induced adverse late effects with respect to the vascular compartment remain elusive, and no causative radioprotective treatment is available to date. RESULTS: The importance of injury to EC for radiation-induced late toxicity in lungs after whole thorax irradiation (WTI) was investigated using a mouse model of radiation-induced pneumopathy. We show that WTI induces EC loss as long-term complication, which is accompanied by the development of fibrosis. Adoptive transfer of mesenchymal stem cells (MSCs) either derived from bone marrow or aorta (vascular wall-resident MSCs) in the early phase after irradiation limited the radiation-induced EC loss and fibrosis progression. Furthermore, MSC-derived culture supernatants rescued the radiation-induced reduction in viability and long-term survival of cultured lung EC. We further identified the antioxidant enzyme superoxide dismutase 1 (SOD1) as a MSC-secreted factor. Importantly, MSC treatment restored the radiation-induced reduction of SOD1 levels after WTI. A similar protective effect was achieved by using the SOD-mimetic EUK134, suggesting that MSC-derived SOD1 is involved in the protective action of MSC, presumably through paracrine signaling. INNOVATION: In this study, we explored the therapeutic potential of MSC therapy to prevent radiation-induced EC loss (late effect) and identified the protective mechanisms of MSC action. CONCLUSIONS: Adoptive transfer of MSCs early after irradiation counteracts radiation-induced vascular damage and EC loss as late adverse effects. The high activity of vascular wall-derived MSCs for radioprotection may be due to their tissue-specific action. Antioxid. Redox Signal. 26, 563-582.

Glycoproteomics Reveals Decorin Peptides With Anti-Myostatin Activity in Human Atrial Fibrillation.

BACKGROUND: Myocardial fibrosis is a feature of many cardiac diseases. We used proteomics to profile glycoproteins in the human cardiac extracellular matrix (ECM). METHODS: Atrial specimens were analyzed by mass spectrometry after extraction of ECM proteins and enrichment for glycoproteins or glycopeptides. RESULTS: ECM-related glycoproteins were identified in left and right atrial appendages from the same patients. Several known glycosylation sites were confirmed. In addition, putative and novel glycosylation sites were detected. On enrichment for glycoproteins, peptides of the small leucine-rich proteoglycan decorin were identified consistently in the flowthrough. Of all ECM proteins identified, decorin was found to be the most fragmented. Within its protein core, 18 different cleavage sites were identified. In contrast, less cleavage was observed for biglycan, the most closely related proteoglycan. Decorin processing differed between human ventricles and atria and was altered in disease. The C-terminus of decorin, important for the interaction with connective tissue growth factor, was detected predominantly in ventricles in comparison with atria. In contrast, atrial appendages from patients in persistent atrial fibrillation had greater levels of full-length decorin but also harbored a cleavage site that was not found in atrial appendages from patients in sinus rhythm. This cleavage site preceded the N-terminal domain of decorin that controls muscle growth by altering the binding capacity for myostatin. Myostatin expression was decreased in atrial appendages of patients with persistent atrial fibrillation and hearts of decorin null mice. A synthetic peptide corresponding to this decorin region dose-dependently inhibited the response to myostatin in cardiomyocytes and in perfused mouse hearts. CONCLUSIONS: This proteomics study is the first to analyze the human cardiac ECM. Novel processed forms of decorin protein core, uncovered in human atrial appendages, can regulate the local bioavailability of antihypertrophic and profibrotic growth factors.