A YAP/TAZ-TEAD signalling module links endothelial nutrient acquisition to angiogenic growth.

Angiogenesis, the process by which endothelial cells (ECs) form new blood vessels from existing ones, is intimately linked to the tissue's metabolic milieu and often occurs at nutrient-deficient sites. However, ECs rely on sufficient metabolic resources to support growth and proliferation. How endothelial nutrient acquisition and usage are regulated is unknown. Here we show that these processes are instructed by Yes-associated protein 1 (YAP)/WW domain-containing transcription regulator 1 (WWTR1/TAZ)-transcriptional enhanced associate domain (TEAD): a transcriptional module whose function is highly responsive to changes in the tissue environment. ECs lacking YAP/TAZ or their transcriptional partners, TEAD1, 2 and 4 fail to divide, resulting in stunted vascular growth in mice. Conversely, activation of TAZ, the more abundant paralogue in ECs, boosts proliferation, leading to vascular hyperplasia. We find that YAP/TAZ promote angiogenesis by fuelling nutrient-dependent mTORC1 signalling. By orchestrating the transcription of a repertoire of cell-surface transporters, including the large neutral amino acid transporter SLC7A5, YAP/TAZ-TEAD stimulate the import of amino acids and other essential nutrients, thereby enabling mTORC1 activation. Dissociating mTORC1 from these nutrient inputs-elicited by the loss of Rag GTPases-inhibits mTORC1 activity and prevents YAP/TAZ-dependent vascular growth. Together, these findings define a pivotal role for YAP/TAZ-TEAD in controlling endothelial mTORC1 and illustrate the essentiality of coordinated nutrient fluxes in the vasculature.

Control of endothelial quiescence by FOXO-regulated metabolites.

Endothelial cells (ECs) adapt their metabolism to enable the growth of new blood vessels, but little is known how ECs regulate metabolism to adopt a quiescent state. Here, we show that the metabolite S-2-hydroxyglutarate (S-2HG) plays a crucial role in the regulation of endothelial quiescence. We find that S-2HG is produced in ECs after activation of the transcription factor forkhead box O1 (FOXO1), where it limits cell cycle progression, metabolic activity and vascular expansion. FOXO1 stimulates S-2HG production by inhibiting the mitochondrial enzyme 2-oxoglutarate dehydrogenase. This inhibition relies on branched-chain amino acid catabolites such as 3-methyl-2-oxovalerate, which increase in ECs with activated FOXO1. Treatment of ECs with 3-methyl-2-oxovalerate elicits S-2HG production and suppresses proliferation, causing vascular rarefaction in mice. Our findings identify a metabolic programme that promotes the acquisition of a quiescent endothelial state and highlight the role of metabolites as signalling molecules in the endothelium.

Involvement of NDPK-B in Glucose Metabolism-Mediated Endothelial Damage via Activation of the Hexosamine Biosynthesis Pathway and Suppression of O-GlcNAcase Activity.

Our previous studies identified that retinal endothelial damage caused by hyperglycemia or nucleoside diphosphate kinase-B (NDPK-B) deficiency is linked to elevation of angiopoietin-2 (Ang-2) and the activation of the hexosamine biosynthesis pathway (HBP). Herein, we investigated how NDPK-B is involved in the HBP in endothelial cells (ECs). The activities of NDPK-B and O-GlcNAcase (OGA) were measured by in vitro assays. Nucleotide metabolism and O-GlcNAcylated proteins were assessed by UPLC-PDA (Ultra-performance liquid chromatography with Photodiode array detection) and immunoblot, respectively. Re-expression of NDPK-B was achieved with recombinant adenoviruses. Our results show that NDPK-B depletion in ECs elevated UDP-GlcNAc levels and reduced NDPK activity, similar to high glucose (HG) treatment. Moreover, the expression and phosphorylation of glutamine:fructose-6-phosphate amidotransferase (GFAT) were induced, whereas OGA activity was suppressed. Furthermore, overall protein O-GlcNAcylation, along with O-GlcNAcylated Ang-2, was increased in NDPK-B depleted ECs. Pharmacological elevation of protein O-GlcNAcylation using Thiamet G (TMG) or OGA siRNA increased Ang-2 levels. However, the nucleoside triphosphate to diphosphate (NTP/NDP) transphosphorylase and histidine kinase activity of NDPK-B were dispensable for protein O-GlcNAcylation. NDPK-B deficiency hence results in the activation of HBP and the suppression of OGA activity, leading to increased protein O-GlcNAcylation and further upregulation of Ang-2. The data indicate a critical role of NDPK-B in endothelial damage via the modulation of the HBP.

A three-gene methylation marker panel for the nodal metastatic risk assessment of muscle-invasive bladder cancer.

PURPOSE: In this study, we aimed to identify a DNA methylation pattern suitable for prognosis assessment of muscle-invasive bladder cancer and to investigate metastasis-associated processes regulated by DNA methylation. METHODS: Genome-wide methylation analysis was performed on 23 muscle-invasive bladder tumors by microarray analysis. Validation was performed by the qAMP technique in two different patient cohorts (n = 32 and n = 100). mRNA expression was analyzed in 12 samples. Protein expression was determined using tissue microarrays of 291 patients. Bladder cancer cell lines T24 and 253JB-V were used for functional analyses. RESULTS: Microarray analyses revealed KISS1R, SEPT9 and CSAD as putative biomarkers with hypermethylation in node-positive tumors. The combination of the three genes predicted the metastatic risk with sensitivity of 73% and specificity of 71% in cohort 1, and sensitivity of 82% and specificity of 54% in cohort 2. mRNA expression differences were detected for KISS1R (p = 0.04). Protein expression of KISS1R was significantly reduced (p < 0.001). Knockdown of SEPT9v3 resulted in increased cell migration by 28% (p = 0.04) and increased invasion by 22% (p = 0.004). KISS1R overexpression resulted in decreased cell migration (25%, p = 0.1). CONCLUSIONS: We identified a methylation marker panel suitable to differentiate between patients with positive and negative lymph nodes at time of cystectomy. This enables a risk assessment for patients who potentially benefit from extended lymph node resection as well as from neoadjuvant chemotherapy and could improve the survival rates. Furthermore, we examined the impact of putative markers on tumor behavior. Hence, KISS1R and SEPT9 could represent a starting point for the development of novel therapy approaches.

Loss of pyruvate kinase M2 limits growth and triggers innate immune signaling in endothelial cells.

Despite their inherent proximity to circulating oxygen and nutrients, endothelial cells (ECs) oxidize only a minor fraction of glucose in mitochondria, a metabolic specialization that is poorly understood. Here we show that the glycolytic enzyme pyruvate kinase M2 (PKM2) limits glucose oxidation, and maintains the growth and epigenetic state of ECs. We find that loss of PKM2 alters mitochondrial substrate utilization and impairs EC proliferation and migration in vivo. Mechanistically, we show that the NF-κB transcription factor RELB is responsive to PKM2 loss, limiting EC growth through the regulation of P53. Furthermore, S-adenosylmethionine synthesis is impaired in the absence of PKM2, resulting in DNA hypomethylation, de-repression of endogenous retroviral elements (ERVs) and activation of antiviral innate immune signalling. This work reveals the metabolic and functional consequences of glucose oxidation in the endothelium, highlights the importance of PKM2 for endothelial growth and links metabolic dysfunction with autoimmune activation in ECs.

FGF-dependent metabolic control of vascular development.

Blood and lymphatic vasculatures are intimately involved in tissue oxygenation and fluid homeostasis maintenance. Assembly of these vascular networks involves sprouting, migration and proliferation of endothelial cells. Recent studies have suggested that changes in cellular metabolism are important to these processes. Although much is known about vascular endothelial growth factor (VEGF)-dependent regulation of vascular development and metabolism, little is understood about the role of fibroblast growth factors (FGFs) in this context. Here we identify FGF receptor (FGFR) signalling as a critical regulator of vascular development. This is achieved by FGF-dependent control of c-MYC (MYC) expression that, in turn, regulates expression of the glycolytic enzyme hexokinase 2 (HK2). A decrease in HK2 levels in the absence of FGF signalling inputs results in decreased glycolysis, leading to impaired endothelial cell proliferation and migration. Pan-endothelial- and lymphatic-specific Hk2 knockouts phenocopy blood and/or lymphatic vascular defects seen in Fgfr1/Fgfr3 double mutant mice, while HK2 overexpression partly rescues the defects caused by suppression of FGF signalling. Thus, FGF-dependent regulation of endothelial glycolysis is a pivotal process in developmental and adult vascular growth and development.

FOXO1 couples metabolic activity and growth state in the vascular endothelium.

Endothelial cells (ECs) are plastic cells that can switch between growth states with different bioenergetic and biosynthetic requirements. Although quiescent in most healthy tissues, ECs divide and migrate rapidly upon proangiogenic stimulation. Adjusting endothelial metabolism to the growth state is central to normal vessel growth and function, yet it is poorly understood at the molecular level. Here we report that the forkhead box O (FOXO) transcription factor FOXO1 is an essential regulator of vascular growth that couples metabolic and proliferative activities in ECs. Endothelial-restricted deletion of FOXO1 in mice induces a profound increase in EC proliferation that interferes with coordinated sprouting, thereby causing hyperplasia and vessel enlargement. Conversely, forced expression of FOXO1 restricts vascular expansion and leads to vessel thinning and hypobranching. We find that FOXO1 acts as a gatekeeper of endothelial quiescence, which decelerates metabolic activity by reducing glycolysis and mitochondrial respiration. Mechanistically, FOXO1 suppresses signalling by MYC (also known as c-MYC), a powerful driver of anabolic metabolism and growth. MYC ablation impairs glycolysis, mitochondrial function and proliferation of ECs while its EC-specific overexpression fuels these processes. Moreover, restoration of MYC signalling in FOXO1-overexpressing endothelium normalizes metabolic activity and branching behaviour. Our findings identify FOXO1 as a critical rheostat of vascular expansion and define the FOXO1-MYC transcriptional network as a novel metabolic checkpoint during endothelial growth and proliferation.

Human endothelial-like differentiated precursor cells maintain their endothelial characteristics when cocultured with mesenchymal stem cell and seeded onto human cancellous bone.

INTRODUCTION: Cancellous bone is frequently used for filling bone defects in a clinical setting. It provides favourable conditions for regenerative cells such as MSC and early EPC. The combination of MSC and EPC results in superior bone healing in experimental bone healing models. MATERIALS AND METHODS: We investigated the influence of osteogenic culture conditions on the endothelial properties of early EPC and the osteogenic properties of MSC when cocultured on cancellous bone. Additionally, cell adhesion, metabolic activity, and differentiation were assessed 2, 6, and 10 days after seeding. RESULTS: The number of adhering EPC and MSC decreased over time; however the cells remained metabolically active over the 10-day measurement period. In spite of a decline of lineage specific markers, cells maintained their differentiation to a reduced level. Osteogenic stimulation of EPC caused a decline but not abolishment of endothelial characteristics and did not induce osteogenic gene expression. Osteogenic stimulation of MSC significantly increased their metabolic activity whereas collagen-1α and alkaline phosphatase gene expressions declined. When cocultured with EPC, MSC's collagen-1α gene expression increased significantly. CONCLUSION: EPC and MSC can be cocultured in vitro on cancellous bone under osteogenic conditions, and coculturing EPC with MSC stabilizes the latter's collagen-1α gene expression.

Simvastatin inhibits cell growth and induces apoptosis and G0/G1 cell cycle arrest in hepatic cancer cells.

Hepatocellular carcinoma (HCC) is one of major health concerns worldwide and one of leading causes of cancer death after lung and gastric cancers. Simvastatin is a cholesterol-lowering drug which inhibits 3-hydroxy-3-methylglutarylcoenzyme CoA (HMG-CoA) reductase. Simvastatin exhibits numerous pleiotropic effects including anti-cancer activity. Yet, the anticancer effects in HCC remain poorly characterized. Therefore, in this study, we investigated the effects of simvastatin on tumor cell growth, apoptosis and cell cycle. HepG2 and Huh7 cell lines were treated with simvastatin (32 and 64 µM) for different time periods. Tumor cell growth was assessed using MTT assay. Apoptosis and cell cycle analysis were also evaluated. Analysis of cell cycle proteins involved in simvastatin-induced manipulation was performed by Western blot and quantitative RT-PCR analyses. Simvastatin induced a reduction of tumor cell growth. In both cell lines, simvastatin induced apoptosis and impaired cell cycle progression as depicted by the greater rates of G0/G1-phase cells than the rates of S-phase cells. Protein expression levels of cell cycle regulating proteins CDK1, CDK2, CDK4, cyclin D1, cyclin E, p19 and p27 were markedly altered by simvastatin. Moreover, CDC2, CCND1 and CDCN2D mRNA expressions were also altered by drug treatment. Collectively, these results suggest that simvastatin induces apoptosis in tumor cells and its anti-proliferative activity was accompanied by inhibition of cyclin-dependent kinases and cyclins, whereas CDK inhibitors p19 and p27 were enhanced. These results may provide novel insights into simvastatin tumor-suppressive action.

Comparison of six bone-graft substitutes regarding to cell seeding efficiency, metabolism and growth behaviour of human mesenchymal stem cells (MSC) in vitro.

INTRODUCTION: Various synthetic bone-graft substitutes are used commercially as osteoconductive scaffolds in the treatment of bone defects and fractures. The role of bone-graft substitutes is changing from osteoconductive conduits for growth to an delivery system for biologic fracture treatments. Achieving optimal bone regeneration requires biologics (e.g. MSC) and using the correct scaffold incorporated into a local environment for bone regeneration. The need for an unlimited supply with high quality bone-graft substitutes continue to find alternatives for bone replacement surgery. MATERIALS AND METHODS: This in vitro study investigates cell seeding efficiency, metabolism, gene expression and growth behaviour of MSC sown on six commercially clinical available bone-graft substitutes in order to define their biological properties: synthetic silicate-substituted porous hydroxyapatite (Actifuse ABX), synthetic alpha-TCP (Biobase), synthetic beta-TCP (Vitoss), synthetic beta-TCP (Chronos), processed human cancellous allograft (Tutoplast) and processed bovines hydroxyapatite ceramic (Cerabone). 250,000 MSC derived from human bone marrow (n=4) were seeded onto the scaffolds, respectively. On days 2, 6 and 10 the adherence of MSC (fluorescence microscopy) and cellular activity (MTT assay) were analysed. Osteogenic gene expression (cbfa-1) was analysed by RT-PCR and scanning electron microscopy was performed. RESULTS: The highest number of adhering cells was found on Tutoplast (e.g. day 6: 110.0+/-24.0 cells/microscopic field; p<0.05) followed by Chronos (47.5+/-19.5, p<0.05), Actifuse ABX (19.1+/-4.4), Biobase (15.7+/-9.9), Vitoss (8.8+/-8.7) and Cerabone (8.1+/-2.2). MSC seeded onto Tutoplast showed highest metabolic activity and gene expression of cbfa-1. These data are confirmed by scanning electron microscopy. The cell shapes varied from round-shaped cells to wide spread cells and cell clusters, depending on the bone-graft substitutes. Processed human cancellous allograft is a well-structured and biocompatible scaffold for ingrowing MSC in vitro. Of all other synthetical scaffolds, beta-tricalcium phosphate (Chronos) have shown the best growth behaviour for MSC. DISCUSSION: Our results indicate that various bone-graft substitutes influence cell seeding efficiency, metabolic activity and growth behaviour of MSC in different manners. We detected a high variety of cellular integration of MSC in vitro, which may be important for bony integration in the clinical setting.

Endothelial progenitor cells and mesenchymal stem cells seeded onto beta-TCP granules enhance early vascularization and bone healing in a critical-sized bone defect in rats.

UNLABELLED: QUESTION/AIM: Lack of vessels indicates an insufficient nutritional supply of a bone graft and may limit the recruitment of bone-forming cells. Our aim was to evaluate the influence of endothelial progenitor cells (EPCs) alone or in combination with mesenchymal stem cells (MSCs) on early vascularization and bone healing in critical-sized defect (CSD) in vivo. METHODS: MSCs from human bone marrow and EPCs from buffy coat were used. A femoral CSD in adult athymic rats was created and stabilized by an external fixateur. The remaining defects were filled with fibronectin-coated beta-tricalcium phosphate (beta-TCP) granules, EPCs seeded on beta-TCP, MSCs seeded on beta-TCP, coculture of EPCs/MSCs seeded on beta-TCP, or autologous bone. Vascularization and bone formation were determined by immunohistology, microCT analysis, and biomechanical testing after 1, 4, and 8 weeks. RESULTS: Early vascularization was significantly improved in EPC/MSC group or EPC group, respectively. At 4 weeks bone formation increased significantly when the CSD was treated with coculture of MSCs/EPCs. Eight weeks after transplantation CSD showed significantly more bony bridgings and significantly increased ultimate load in the EPC/MSC group compared to the other groups. DISCUSSION: This cell approach suggests that there is a synergistic effect and that the initial stage of neovascularization by EPCs is considered to be crucial for complete bone regeneration in the late phase.

Simultaneous cultivation of human endothelial-like differentiated precursor cells and human marrow stromal cells on beta-tricalcium phosphate.

AIM: The size of a bone defect limits the ingrowth of bone-forming cells. Endothelial cell-like differentiated precursor cells (endothelial progenitor cells, EPC) enhance the neovascularization, while marrow stromal cells (MSC) promote the repair of bone defects. Our aim was to evaluate if both types of cells can be cocultivated on a beta-tricalcium phosphate (beta-TCP) matrix and maintain their differentiation capacity as well as to analyze the biologic activity of these cell constructs in vivo. METHODS: MSC from human bone marrow and EPC from buffy coat were used. EPC and MSC, alone or in combination, were seeded on fibronectin-coated beta-TCP. After 2, 6, and 10 days the metabolic activity and the endothelial differentiation were tested. On day 10 real-time RT-PCRs for endothelial genes (von Willebrandt factor, vascular endothelial growth factor, and vascular endothelial growth factor-receptor 2), osteogenic genes (osteocalcin, cbfa-1, and collagen-1alpha), and the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase were performed. Cell-containing constructs were implanted into the critical-size defect of the femur of the nude rat. Bone formation and vascularization was determined after 1 week. RESULTS: MSC and EPC on beta-TCP remain metabolically active over 10 days. They maintain their differentiation as measured by means of Dil-ac-LDL uptake (EPC) and gene expression of lineage typical genes (EPC + MSC). Although a potential osteogenic differentiation of MSC was maybe affected negatively, constructs loaded with MSC resulted in an increase of new bone mass. Constructs containing EPC resulted in an improved vasculogenesis in vivo. DISCUSSION: MSC and EPC can be cultivated in combination on a fibronectin-coated beta-TCP, thereby partly maintaining their lineage typical gene expression. The results of the in vivo examinations suggest that beta-TCP combined with EPC and MSC can used as a suitable tool to foster bone healing.

A Hepatozoon species genetically distinct from H. canis infecting spotted hyenas in the Serengeti ecosystem, Tanzania.

Health monitoring of spotted hyenas (Crocuta crocuta) in the Serengeti ecosystem, Tanzania, revealed Hepatozoon infection in all of 11 immature individuals examined following death from natural causes. Hepatozoon infection was probably an important factor contributing to mortality in two cases that exhibited clinical signs of ataxia, lethargy, ocular discharge, retching, and labored breathing before death. Whether Hepatozoon infection contributed to six deaths from fire, probable lion predation and unknown causes could not be determined. Four deaths from infanticide and starvation were unlikely to be associated with Hepatozoon infection. Histologic examination revealed lung tissue infected with cyst-like structures containing protozoan stages in all eight cases examined and interstitial pneumonia in most cases. Systemic spread of infection to several organs was found in three cases. Alignment of a 426 bp sequence from the parasite's 18s rRNA gene revealed a Hepatozoon species identical to that recently described from two domestic cats in Spain and only 7 bp substitutions when a 853 bp sequence was aligned to this cat Hepatozoon species. Previous reports of infection of wild carnivores in eastern and southern Africa with an unspecified Hepatozoon species similar in appearance to H. canis may have involved the species described in this study.

High dosage of simvastatin reduces TNF-alpha-induced apoptosis of endothelial progenitor cells but fails to prevent apoptosis induced by IL-1beta in vitro.

Endothelial progenitor cells (EPC) could provide a possible source for the improvement of neovascularization in injured tissues following multiple trauma. Recently, it became obvious that at least two types of EPC can be cultured from peripheral blood mononuclear cells. In this work we focused on the fraction of the easily accessible early EPC, which can be generated in clinically relevant amounts within 5 days. Periods of hyper-inflammation, systemic or local, often occur during a multiple trauma. Thus, this study was conducted to elucidate the influence of the prototypical proinflammatory cytokines interleukin (IL)-1beta and tumor necrosis factor-alpha (TNF-alpha) on the survival of early EPC. In the past years it was observed that HMG-CoA reductase inhibitors (statins) exert protective effects during inflammatory processes. Therefore, the effect of a preconditioning of early EPC with simvastatin on the survival of EPC under proinflammatory conditions was tested as well. Incubation with 50 mu/mL TNF-alpha [0.45 ng/mL] or IL-1beta [0.25 ng/mL] resulted in a 3-fold (18.4 +/- 2.9%), respectively, 4-fold (25.5 +/- 3.4%) increase of apoptotic EPC in comparison to the untreated control (6.1 +/- 1.6%). In accordance, 24 h after the cytokines had been added, the EPC number per high power field decreased significantly. A preconditioning with simvastatin [25 microM] resulted in significant inhibition of the TNF-alpha-induced apoptosis, whereas the IL-1beta-mediated apoptosis was only slightly reduced. In conclusion, this study shows clearly that TNF-alpha and IL-1beta are harmful to early EPC and that the HMG-CoA reductase inhibitor simvastatin protects EPC from TNF-alpha- and eventually from IL-1beta-mediated apoptosis. These results suggest that simvastatin has protective effects on EPC survival and differentiation in a hyperinflammatory situation.

Interleukin 8 mRNA gene expression in peripheral and intra-abdominal neutrophils during human secondary peritonitis.

The aim of the study was to investigate interleukin 8 (IL-8) mRNA gene expression in circulating and emigrated intra-abdominal neutrophils during human secondary peritonitis intra- and postoperatively (until 96 h). Patients with secondary peritonitis were allocated to two groups, e.g., patients with no complications (n = 10) and patients with complications (organ failure, septic shock, etc., n = 9). Patients with elective abdominal surgery (n = 11) and a group with healthy volunteers (n = 7) were studied as controls. Neutrophil RNA was isolated and semiquantitative reverse transcription-PCR was performed. The PCR products were compared with corresponding GAPDH bands (=100%). The highest amount of IL-8 mRNA could be assessed in blood neutrophils of healthy volunteers (87.4% +/- 7.4%). Complicated peritonitis was associated with the lowest concentration of IL-8 mRNA in blood neutrophils intraoperatively (24% +/- 7%, P < 0.05), which showed no recovery throughout the observation period (34% +/- 8%, 96 h postoperatively). IL-8 mRNA concentration in blood neutrophils of patients with uncomplicated peritonitis and patients with elective abdominal surgery was higher intraoperatively (55.2% +/- 9% (uncomplicated peritonitis); 68% +/- 15% (elective abdominal surgery, P < 0.05 versus complicated peritonitis). Thereafter, IL-8 mRNA decreased slightly in both groups, but was distinctly higher than in patients with complicated peritonitis. Emigration to the abdominal cavity resulted in an approximately 2-fold, in some cases 3-fold, increase in the concentration of IL-8 mRNA in emigrated intra-abdominal neutrophils when compared with circulating cells. This increase could be observed in all groups. The long-lasting down-regulation of constitutive gene expression of IL-8 mRNA in blood neutrophils during complicated peritonitis is worrying because IL-8 is an important activator and chemoattractant for neutrophils themselves. It is encouraging that migration to another compartment, e.g., infected abdominal cavity, resulted in an increase in neutrophil IL-8 mRNA during complicated and uncomplicated peritonitis.

Serum derived from multiple trauma patients promotes the differentiation of endothelial progenitor cells in vitro: possible role of transforming growth factor-beta1 and vascular endothelial growth factor165.

Ischemia in various organs and tissues takes place during and as a direct result of multiple trauma (MT). Bone marrow-derived endothelial progenitor cells (EPCs) are involved in neovascularization after ischemic incidences. Here, we report that serum derived from patients with MT stimulates differentiation of EPCs in vitro from peripheral blood mononuclear cells (PBMCs). EPCs were identified by DiL-Acetyl-LDL-uptake with concomitant UEA-I-lectin binding. A significant increase in EPC numbers was noted when PBMCs were cultivated for 72 h with the serum of MT patients (n = 25) obtained at 5 days. Furthermore, serum from MT patients enhanced the functional acting of EPCs to form prevascular structures in matrigel. Reverse transcription polymerase chain reaction analysis revealed gene expression of transforming growth factor (TGF)-beta1- and vascular endothelial growth factor (VEGF) receptors 1 and 2. Reverse transcription polymerase chain reaction analysis was based on further cultivated cell preparations, which contained at least 80% EPCs. Moreover, the addition of recombinant VEGF or low concentrations of TGF-beta increased EPC differentiation. In addition, neutralization of TGF-beta1 and of VEGF165 in MT serum using specific antibodies resulted in a significant decrease in EPC differentiation. Our data indicate that TGF-beta1 and VEGF165 play a pivotal role for EPC differentiation induced by serum of polytrauma patients.

Phagocytosis by emigrated, intra-abdominal neutrophils is depressed during human secondary peritonitis.

The phagocytic function of neutrophils is a crucial element in host defense against invading microorganisms. Patients with diffuse peritonitis depend on adequate reactivity of neutrophils, in particular locally in the peritoneal cavity as well as in the circulation. This study examined phagocytosis as well as numerical expression of Fcgamma I-III (CD16, CD32, CD64) and complement receptors (CD18, CD35) of emigrated, intra-abdominal and circulating neutrophils during human secondary peritonitis using fluorescence-activated cell analysis. Optimally opsonized E. coli bacteria were used independently of the well-known low level of opsonic molecules during peritonitis. Compared with controls (abdominal surgery without peritonitis), the percentage of emigrated neutrophils which engulfed E. coli bacteria was significantly depressed until 48 h after diagnosis of, and surgery for, peritonitis. When patients with complicated peritonitis (septic shock, multiple organ failure) were compared with patients without complications, phagocytosis was even more depressed in patients with complications. Numerical expression of CD64 (Fcgamma RI) and CD35 (CR1) increased significantly on emigrated polymorphonuclear leukocytes (PMNs) during peritonitis when compared to controls. There was no difference in CD18 and CD32 (Fcgamma RII) expression between the two groups. Numerical expression of CD16 (Fcgamma RIII) on emigrated PMNs decreased significantly in peritonitis. This was more pronounced in patients with complicated peritonitis. We conclude that there is a long-lasting depression of phagocytosis by emigrated PMNs during peritonitis, independent of the opsonic activity. Our data suggest that decreased phagocytosis might be correlated to the profound drop in CD16 on these cells.