Validation of CSF-1 receptor (CD115) staining for analysis of murine monocytes by flow cytometry.

CD115, the receptor for colony stimulating factor 1, is essential for survival and differentiation of monocytes and macrophages and is therefore frequently used to define monocyte subsets and their progenitors in immunological assays. However, CD115 surface expression and detection by flow cytometry is greatly influenced by cell isolation and processing methods, organ source, and disease context. In a systematic analysis of murine monocytes, we define experimental conditions that preserve or limit CD115 surface expression and staining by flow cytometry. We also find that, independent of conditions, CD115 surface levels are consistently lower in Ly6Clo monocytes than in Ly6Chi monocytes, with the exception of Ly6Clo monocytes in the bone marrow. Furthermore, in contrast to IL-34, the presence of colony stimulating factor 1 impairs CD115 antibody staining in a dose-dependent manner, which, in a model of ischemic kidney injury with elevated levels of colony stimulating factor 1, influenced quantification of kidney monocytes. Thus, staining and experimental conditions affect quantitative and qualitative analysis of monocytes and may influence experimental conclusions.

CSF-1 and Notch signaling cooperate in macrophage instruction and tissue repair during peripheral limb ischemia.

Ischemia causes an inflammatory response featuring monocyte-derived macrophages (MF) involved in angiogenesis and tissue repair. Angiogenesis and ischemic macrophage differentiation are regulated by Notch signaling via Notch ligand Delta-like 1 (Dll1). Colony stimulating factor 1 (CSF-1) is an essential MF lineage factor, but its role in ischemic macrophage development and the interaction with Notch signaling is so far unclear. Using a mouse model of hind limb ischemia with CSF-1 inhibitor studies and Dll1 heterozygous mice we show that CSF-1 is induced in the ischemic niche by a subpopulation of stromal cells expressing podoplanin, which was paralleled by the development of ischemic macrophages. Inhibition of CSF-1 signaling with small molecules or blocking antibodies impaired macrophage differentiation but prolonged the inflammatory response, resulting in impaired perfusion recovery and tissue regeneration. Yet, despite high levels of CSF-1, macrophage maturation and perfusion recovery were impaired in mice with Dll1 haploinsufficiency, while inflammation was exaggerated. In vitro, CSF-1 was not sufficient to induce full MF differentiation from donor monocytes in the absence of recombinant DLL1, while the presence of DLL1 in a dose-dependent manner stimulated MF differentiation in combination with CSF-1. Thus, CSF-1 is an ischemic niche factor that cooperates with Notch signaling in a non-redundant fashion to instruct macrophage cell fate and maturation, which is required for ischemic perfusion recovery and tissue repair.

A new fasciocutaneous flap model identifies a critical role for endothelial Notch signaling in wound healing and flap survival.

Flap surgery is a common treatment for severe wounds and a major determinant of surgical outcome. Flap survival and healing depends on adaptation of the local flap vasculature. Using a novel and defined model of fasciocutaneous flap surgery, we demonstrate that the Notch ligand Delta-like 1 (Dll1), expressed in vascular endothelial cells, regulates flap arteriogenesis, inflammation and flap survival. Utilizing the stereotyped anatomy of dorsal skin arteries, ligation of the major vascular pedicle induced strong collateral vessel development by end-to-end anastomosis in wildtype mice, which supported flap perfusion recovery over time. In mice with heterozygous deletion of Dll1, collateral vessel formation was strongly impaired, resulting in aberrant vascularization and subsequent necrosis of the tissue. Furthermore, Dll1 deficient mice showed severe inflammation in the flap dominated by monocytes and macrophages. This process is controlled by endothelial Dll1 in vivo, since the results were recapitulated in mice with endothelial-specific deletion of Dll1. Thus, our model provides a platform to study vascular adaptation to flap surgery and molecular and cellular regulators influencing flap healing and survival.

High phosphate-induced progressive proximal tubular injury is associated with the activation of Stat3/Kim-1 signaling pathway and macrophage recruitment.

Dietary phosphate intake in the Western population greatly exceeds the recommended dietary allowance and is linked to enhanced cardiovascular and all-cause mortality. It is unclear whether a chronic high phosphate diet (HPD) causes kidney injury in healthy individuals. Here, we show that feeding a 2% HPD in C57BL/6N mice for one up to six months resulted in hyperphosphatemia, hyperphosphaturia, increased plasma levels of fibroblast growth factor (FGF) 23, and parathyroid hormone (PTH) compared to mice on a 0.8% phosphate diet. Kidney injury was already noted after two months of HPD characterized by loss of proximal tubular (PT) cell polarity, flattened epithelia, disruption of brush border membranes, vacuolization, increased PT cell proliferation, marked interstitial mononuclear infiltration, and progressive accumulation of collagen fibers. HPD increased Stat3 activation and Kim-1 expression in PT epithelial cells and enhanced renal synthesis of chemokines recruiting monocytes and macrophages as well as macrophage related factors. Enhanced recruitment of F4/80+ macrophages around injured PT lesions was timely associated with increased Kim-1 synthesis, tubular MCP-1 expression, and degree of PT injury score. Likewise, tubulointerstitial fibrosis was associated with activation of Stat3/Kim-1 signaling pathway. The stimulation of human proximal tubular cells with high phosphate activated Stat3 signaling and induced HAVCR1 and CCL2 expression. We conclude that high phosphate results in progressive proximal tubular injury, indicating that high dietary phosphate intake may affect kidney health and therefore represents an underestimated health problem for the general population.

Blood vessel control of macrophage maturation promotes arteriogenesis in ischemia.

Ischemia causes an inflammatory response that is intended to restore perfusion and homeostasis yet often aggravates damage. Here we show, using conditional genetic deletion strategies together with adoptive cell transfer experiments in a mouse model of hind limb ischemia, that blood vessels control macrophage differentiation and maturation from recruited monocytes via Notch signaling, which in turn promotes arteriogenesis and tissue repair. Macrophage maturation is controlled by Notch ligand Dll1 expressed in vascular endothelial cells of arteries and requires macrophage canonical Notch signaling via Rbpj, which simultaneously suppresses an inflammatory macrophage fate. Conversely, conditional mutant mice lacking Dll1 or Rbpj show proliferation and transient accumulation of inflammatory macrophages, which antagonizes arteriogenesis and tissue repair. Furthermore, the effects of Notch are sufficient to generate mature macrophages from monocytes ex vivo that display a stable anti-inflammatory phenotype when challenged with pro-inflammatory stimuli. Thus, angiocrine Notch signaling fosters macrophage maturation during ischemia.Molecular mechanisms of macrophage-mediated regulation of artery growth in response to ischemia are poorly understood. Here the authors show that vascular endothelium controls macrophage maturation and differentiation via Notch signaling, which in turn promotes arteriogenesis and ischemic tissue recovery.

Regulation of monocyte cell fate by blood vessels mediated by Notch signalling.

A population of monocytes, known as Ly6C(lo) monocytes, patrol blood vessels by crawling along the vascular endothelium. Here we show that endothelial cells control their origin through Notch signalling. Using combinations of conditional genetic deletion strategies and cell-fate tracking experiments we show that Notch2 regulates conversion of Ly6C(hi) monocytes into Ly6C(lo) monocytes in vivo and in vitro, thereby regulating monocyte cell fate under steady-state conditions. This process is controlled by Notch ligand delta-like 1 (Dll1) expressed by a population of endothelial cells that constitute distinct vascular niches in the bone marrow and spleen in vivo, while culture on recombinant DLL1 induces monocyte conversion in vitro. Thus, blood vessels regulate monocyte conversion, a form of committed myeloid cell fate regulation.

Activation of endothelial ß-catenin signaling induces heart failure.

Activation of ß-catenin-dependent canonical Wnt signaling in endothelial cells plays a key role in angiogenesis during development and ischemic diseases, however, other roles of Wnt/ß-catenin signaling in endothelial cells remain poorly understood. Here, we report that sustained activation of ß-catenin signaling in endothelial cells causes cardiac dysfunction through suppressing neuregulin-ErbB pathway in the heart. Conditional gain-of-function mutation of ß-catenin, which activates Wnt/ß-catenin signaling in Bmx-positive arterial endothelial cells (Bmx/CA mice) led to progressive cardiac dysfunction and 100% mortality at 40 weeks after tamoxifen treatment. Electron microscopic analysis revealed dilatation of T-tubules and degeneration of mitochondria in cardiomyocytes of Bmx/CA mice, which are similar to the changes observed in mice with decreased neuregulin-ErbB signaling. Endothelial expression of Nrg1 and cardiac ErbB signaling were suppressed in Bmx/CA mice. The cardiac dysfunction of Bmx/CA mice was ameliorated by administration of recombinant neuregulin protein. These results collectively suggest that sustained activation of Wnt/ß-catenin signaling in endothelial cells might be a cause of heart failure through suppressing neuregulin-ErbB signaling, and that the Wnt/ß-catenin/NRG axis in cardiac endothelial cells might become a therapeutic target for heart failure.

MAP-Kinase Activated Protein Kinase 2 Links Endothelial Activation and Monocyte/macrophage Recruitment in Arteriogenesis.

Arteriogenesis, the growth of natural bypass arteries, is triggered by hemodynamic forces within vessels and requires a balanced inflammatory response, involving induction of the chemokine MCP-1 and recruitment of leukocytes. However, little is known how these processes are coordinated. The MAP-kinase-activated-proteinkinase-2 (MK2) is a critical regulator of inflammatory processes and might represent an important link between cytokine production and cell recruitment during postnatal arteriogenesis. Therefore, the present study investigated the functional role of MK2 during postnatal arteriogenesis. In a mouse model of hindlimb ischemia (HLI) MK2-deficiency (MK2KO) significantly impaired ischemic blood flow recovery and growth of collateral arteries as well as perivascular recruitment of mononuclear cells and macrophages. This was accompanied by induction of endothelial MCP-1 expression in wildtype (WT) but not in MK2KO collateral arteries. Following HLI, MK2 activation rapidly occured in the endothelium of growing WT arteries in vivo. In vitro, inflammatory cytokines and cyclic stretch activated MK2 in endothelial cells, which was required for stretch- and cytokine-induced release of MCP-1. In addition, a monocyte cell autonomous function of MK2 was uncovered potentially regulating MCP-1-dependent monocyte recruitment to vessels: MCP-1 stimulation of WT monocytes induced MK2 activation and monocyte migration in vitro. The latter was reduced in MK2KO monocytes, while in vivo MK2 was activated in monocytes recruited to collateral arteries. In conclusion, MK2 regulates postnatal arteriogenesis by controlling vascular recruitment of monocytes/macrophages in a dual manner: regulation of endothelial MCP-1 expression in response to hemodynamic and inflammatory forces as well as MCP-1 dependent monocyte migration.

Overexpression of integrin beta 5 enhances the paracrine properties of circulating angiogenic cells via Src kinase-mediated activation of STAT3.

OBJECTIVE: To determine the intracellular mechanisms mediating the angiogenic effects of integrin alpha v beta 5 overexpression in circulating angiogenic cells (CACs). METHODS AND RESULTS: Integrin alpha v beta 5 is expressed on angiogenic endothelial cells, and integrin alpha v beta 5 activation was shown to improve the reparative functions of endothelial progenitors within the cardiovascular system. CACs were transiently transfected with the full-length cDNA of human integrin beta 5 (CAC-ITGB5) or control-vector (CAC-vector). Integrin beta 5 overexpression was confirmed using flow cytometry, Western blot, and PCR analysis; it enhanced the angiogenic capacities of CACs in vitro (spheroid and Matrigel angiogenesis assay) and stimulated new vessel formation in vivo (murine hind limb ischemia model). Overexpression of ITGB5 resulted in integrin alpha v beta 5 phosphorylation and activation of Src kinase and signal transducer and activator of transcription (STAT) 3. Furthermore, elevated mRNA and protein expression of the CXC chemokine CXCL8 and the CC chemokine CCL2 was detected in CAC-ITGB5, and conditioned medium from CAC-ITGB5 enhanced the sprouting of coincubated human endothelial cells in a STAT3-, CXCL8-, and CCL2-dependent manner. CONCLUSIONS: Src kinase-mediated activation of STAT3 and subsequent angiogenic gene expression mediate the effects of integrin alpha v beta 5 and may be exploited to enhance the paracrine activities of CACs.

Ex vivo expanded hematopoietic progenitor cells improve cardiac function after myocardial infarction: role of beta-catenin transduction and cell dose.

Cell-based therapy after myocardial infarction (MI) is a promising therapeutic option but the relevant cell subsets and dosage requirements are poorly defined. We hypothesized that cell therapy for myocardial infarction is improved by ex vivo expansion and high-dose transplantation of defined hematopoietic progenitor cells (HPCs). Since beta-catenin promotes self-renewal of stem cells we evaluated the therapeutic efficacy of beta-catenin-mediated ex vivo expansion of mouse HPCs in a mouse model of myocardial ischemia/reperfusion followed by intraarterial cell delivery. The impact of cell dose was determined by comparing a low-dose (LD, 5 x 10(5) cells) vs. a high-dose (HD, 1 x 10(7) cells) cell transplantation regimen of beta-catenin-HPCs. The impact of beta-catenin modification of HPCs was determined by comparing control-transduced HPCs (GFP-HPCs) vs. transgenic beta-catenin-HPCs. HD beta-catenin-HPCs significantly improved LV function and end-systolic and end-diastolic dimensions as compared to saline and LD beta-catenin-HPCs. Furthermore, while treatment with HD GFP-HPC resulted in a modest cardiac improvement the application of beta-catenin-HPCs was superior, resulting in a significant improvement in EF, FS and LVESD over saline and control GFP-HPC treatment. Although myocardial engraftment of HPCs was only transient, as determined by cell quantification after dye labeling, beta-catenin-HPC treatment significantly decreased infarct size, reduced cardiomyocyte apoptosis and increased capillary angiogenesis in vitro and in vivo. Ex vivo expanded HPCs improve cardiac function and remodeling post MI in a cell number- and beta-catenin-dependent manner.

Haeme oxygenase promotes progenitor cell mobilization, neovascularization, and functional recovery after critical hindlimb ischaemia in mice.

AIMS: Neovascularization is an important element of long-term functional recovery during chronic ischaemia. We postulated that haeme oxygenase (HO) is required for progenitor cell recruitment, neovascularization, and blood flow recovery after critical hindlimb ischaemia (HLI). METHODS AND RESULTS: The femoral artery was ligated in FVB/N mice proximal to its superficial and deep branches. Blood flow in the ischaemic hindlimb was determined by laser Doppler perfusion imaging. Capillary density was measured by isolectin staining, and mobilization of Sca-1(+)/Kdr(+) progenitor cells by FACS analysis. Progenitor cell recruitment to the ischaemic hindlimb was assessed after Tie2-lacZ transgenic bone marrow transplantation. Blood flow recovery after femoral artery ligation was significantly blunted in mice treated with the HO inhibitor tin protoporphyrin-IX (25 mg/kg i.p., every other day). HO-inhibited mice developed more pronounced limb necrosis, associated with impaired hindlimb motor function. Capillary density in the ischaemic hindlimb and mobilization of Sca-1(+)/Kdr(+) progenitor cells were significantly reduced after HO inhibition. After transplantation of Tie2-lacZ transgenic bone marrow cells into lethally irradiated wild-type mice, fewer LacZ(+) cells were detected in the ischaemic hindlimb muscle of HO-inhibited mice. Mechanistically, HO inhibition prevented the establishment of a stromal cell-derived factor-1 gradient for progenitor cell mobilization between the ischaemic hindlimb and bone marrow. CONCLUSION: HOs are required for progenitor cell recruitment, neovascularization, and functional recovery after HLI.

Notch ligand Delta-like 1 is essential for postnatal arteriogenesis.

Growth of functional arteries is essential for the restoration of blood flow to ischemic organs. Notch signaling regulates arterial differentiation upstream of ephrin-B2 during embryonic development, but its role during postnatal arteriogenesis is unknown. Here, we identify the Notch ligand Delta-like 1 (Dll1) as an essential regulator of postnatal arteriogenesis. Dll1 expression was specifically detected in arterial endothelial cells, but not in venous endothelial cells or capillaries. During ischemia-induced arteriogenesis endothelial Dll1 expression was strongly induced, Notch signaling activated and ephrin-B2 upregulated, whereas perivascular cells expressed proangiogenic vascular endothelial growth factor, and the ephrin-B2 activator EphB4. In heterozygous Dll1 mutant mice endothelial Notch activation and ephrin-B2 induction after hindlimb ischemia were absent, arterial collateral growth was abrogated and recovery of blood flow was severely impaired, but perivascular vascular endothelial growth factor and EphB4 expression was unaltered. In vitro, angiogenic growth factors synergistically activated Notch signaling by induction of Dll1, which was necessary and sufficient to regulate ephrin-B2 expression and to induce ephrin-B2 and EphB4-dependent branching morphogenesis in human arterial EC. Thus, Dll1-mediated Notch activation regulates ephrin-B2 expression and postnatal arteriogenesis.

Critical role of endothelial Notch1 signaling in postnatal angiogenesis.

Notch receptors are important mediators of cell fate during embryogenesis, but their role in adult physiology, particularly in postnatal angiogenesis, remains unknown. Of the Notch receptors, only Notch1 and Notch4 are expressed in vascular endothelial cells. Here we show that blood flow recovery and postnatal neovascularization in response to hindlimb ischemia in haploinsufficient global or endothelial-specific Notch1(+/-) mice, but not Notch4(-/-) mice, were impaired compared with wild-type mice. The expression of vascular endothelial growth factor (VEGF) in response to ischemia was comparable between wild-type and Notch mutant mice, suggesting that Notch1 is downstream of VEGF signaling. Treatment of endothelial cells with VEGF increases presenilin proteolytic processing, gamma-secretase activity, Notch1 cleavage, and Hes-1 (hairy enhancer of split homolog-1) expression, all of which were blocked by treating endothelial cells with inhibitors of phosphatidylinositol 3-kinase/protein kinase Akt or infecting endothelial cells with a dominant-negative Akt mutant. Indeed, inhibition of gamma-secretase activity leads to decreased angiogenesis and inhibits VEGF-induced endothelial cell proliferation, migration, and survival. Overexpression of the active Notch1 intercellular domain rescued the inhibitory effects of gamma-secretase inhibitors on VEGF-induced angiogenesis. These findings indicate that the phosphatidylinositol 3-kinase/Akt pathway mediates gamma-secretase and Notch1 activation by VEGF and that Notch1 is critical for VEGF-induced postnatal angiogenesis. These results suggest that Notch1 may be a novel therapeutic target for improving angiogenic response and blood flow recovery in ischemic limbs.

Transcoronary delivery of bone marrow cells to the infarcted murine myocardium: feasibility, cellular kinetics, and improvement in cardiac function.

Efficient strategies for labelling and delivery of bone marrow derived stem cells (BMCs) are required to elucidate the cellular kinetics and therapeutic effects after BMC transfer for myocardial infarction (MI). Lineage negative (lin-) BMCs, labelled ex vivo in a simple procedure with the cell tracker dye tetramethyl-rhodamine (TAMRA), were reliably detected by fluorescence microscopy with higher specificity than retroviral enhanced green fluorescence protein (EGFP) marking and detection. Only few cells entered the ischemic myocardium after intravenous (i.v.) application, but this number increased more than 18-fold after transcoronary delivery. Time course and kinetic analysis over 12 h revealed that myocardial colonization seems to be a biphasic process of first order decay with different elimination half-lives. Most cells are eliminated rapidly during the first 2 h (t1/2 40 min), but the remaining cells are retained significantly longer in the ischemic heart (t1/2 5.2 h). In contrast, BMC colonization of the spleen increased rather in a linear fashion. Although transcoronary BMC transfusion did not alter infarct size, it increased capillary density in the infarct border zone and improved LV function 4 weeks after MI. In conclusion, BMCs delivered by transcoronary injection increase capillary density and improve LV function after MI although homing to the ischemic heart is only transient.

Haematopoietic stem cells improve cardiac function after infarction without permanent cardiac engraftment.

BACKGROUND: Transplantation of bone marrow derived adult stem cells (BMC) improves cardiac function after acute myocardial infarction (MI). However, the cell population mediating myocardial recovery and the fate of the transplanted cells are still controversial. AIMS: We determined the effects of Sca-1+ c-kit+ lin- haematopoietic BMC on cardiac function after MI and the cell fate after transplantation. METHODS: Sca-1+ c-kit+ lin- BMC of male donor C57BL/6 mice were transplanted by intravenous injection into syngenic females after permanent MI. LV dimensions and function were determined by echocardiography and cardiac magnetic resonance imaging, transplanted BMC were identified by Y chromosome DNA in situ hybridization. RESULTS: BMC treatment completely prevented LV dilation (LV end-diastolic volume BMC 70 +/- 16 microl vs. control 122 +/- 41 microl; p < 0.05) and improved fractional shortening (BMC 22.9 +/- 8% vs. control 15.4 +/- 8.4%; p < 0.05) and ejection fraction BMC 68.2 +/- 6.6% vs. control 52 +/- 14.3%; p < 0.05) as early as 3 days after transplantation, but did not decrease infarct size (BMC 27 +/- 6% vs. control 28 +/- 7%, p = n.s.). After 4 weeks, only sporadic cells of male origin were identified in infarcted hearts (< 0.01% of periinfarct cells). CONCLUSION: Intravenous injection of Sca-1+ c-kit+ lin- in BMC after MI improves LV dimensions and function without evidence for long term engraftment.

Essential role of endothelial Notch1 in angiogenesis.

BACKGROUND: Notch signaling influences binary cell fate decisions in a variety of tissues. The Notch1 receptor is widely expressed during embryogenesis and is essential for embryonic development. Loss of global Notch1 function results in early embryonic lethality, but the cell type responsible for this defect is not known. Here, we identify the endothelium as the primary target tissue affected by Notch1 signaling. METHODS AND RESULTS: We generated an endothelium-specific deletion of Notch1 using Tie2Cre and conditional Notch1(flox/flox) mice. Mutant embryos lacking endothelial Notch1 died at approximately embryonic day 10.5 with profound vascular defects in placenta, yolk sac, and embryo proper, whereas heterozygous deletion had no effect. In yolk sacs of mutant embryos, endothelial cells formed a primary vascular plexus indicative of intact vasculogenesis but failed to induce the secondary vascular remodeling required to form a mature network of well-organized large and small blood vessels, which demonstrates a defect in angiogenesis. These vascular defects were also evident in the placenta, where blood vessels failed to invade the placental labyrinth, and in the embryo proper, where defective blood vessel maturation led to pericardial and intersomitic hemorrhage. Enhanced activation of caspase-3 was detected in endothelial and neural cells of mutant mice, which resulted in enhanced apoptotic degeneration of somites and the neural tube. CONCLUSIONS: These findings recapitulate the vascular phenotype of global Notch1-/- mutants and indicate an essential cell-autonomous role of Notch1 signaling in the endothelium during vascular development. These results may have important clinical implications with regard to Notch1 signaling in adult angiogenesis.

Nontranscriptional actions of the glucocorticoid receptor.

Many cellular responses to corticosteroids involve the transcriptional modulation of target genes by a prototypical nuclear receptor, the glucocorticoid receptor (GR). In the classic model of steroid hormone action GR acts as ligand-dependent transcription factor by either activating or repressing gene expression through direct interactions with DNA or other transcription factors. Recent evidence suggests an important role for nontranscriptional effects of GR in the vascular system. The nontranscriptional actions of GR involve the rapid activation of protein kinases, such as phosphatidylinositol-3 kinase and Akt, leading to the activation of endothelial nitric oxide synthase. This novel pathway of steroid hormone action protects against ischemic injury by augmenting blood flow and decreasing vascular inflammation.