Outer membrane porin M35 of Moraxella catarrhalis mediates susceptibility to aminopenicillins

BACKGROUND: The outer membrane protein M35 is a conserved porin of type 1 strains of the respiratory pathogen Moraxella catarrhalis. It was previously shown that M35 is involved in the uptake of essential nutrients required for bacterial growth and for nasal colonization in mice. The aim of this study was (i) to characterize the potential roles of M35 in the host-pathogen interactions considering the known multifunctionality of porins and (ii) to characterize the degree of conservation in the phylogenetic older subpopulation (type 2) of M. catarrhalis. RESULTS: Isogenic m35 mutants of the type 1 strains O35E, 300 and 415 were tested for their antimicrobial susceptibility against 15 different agents. Differences in the MIC (Minimum Inhibitory Concentration) between wild-type and mutant strains were found for eight antibiotics. For ampicillin and amoxicillin, we observed a statistically significant 2.5 to 2.9-fold MIC increase (p < 0.03) in the m35 mutants. Immunoblot analysis demonstrated that human saliva contains anti-M35 IgA. Wild-type strains and their respective m35 mutants were indistinguishable with respect to the phenotypes of autoagglutination, serum resistance, iron acquisition from human lactoferrin, adherence to and invasion of respiratory tract epithelial cells, and proinflammatory stimulation of human monocytes. DNA sequencing of m35 from the phylogenetic subpopulation type 2 strain 287 revealed 94.2% and 92.8% identity on the DNA and amino acid levels, respectively, in comparison with type 1 strains. CONCLUSION: The increase in MIC for ampicillin and amoxicillin, respectively, in the M35-deficient mutants indicates that this porin affects the outer membrane permeability for aminopenicillins in a clinically relevant manner. The presence of IgA antibodies in healthy human donors indicates that M35 is expressed in vivo and recognized as a mucosal antigen by the human host. However, immunoblot analysis of human saliva suggests the possibility of antigenic variation of immunoreactive epitopes, which warrants further analysis before M35 can be considered a potential vaccine candidate.

Identification of Francisella tularensis cluster in central and western Europe

We conducted a molecular analysis of Francisella tularensis strains isolated in Switzerland and identified a specific subpopulation belonging to a cluster of F. tularensis subsp. holarctica that is widely dispersed in central and western continental Europe. This subpopulation was present before the tularemia epidemics on the Iberian Peninsula.

Expression of trefoil factor 1 in the developing and adult rat ventral mesencephalon

Trefoil factor 1 (TFF1) belongs to a family of secreted peptides with a characteristic tree-looped trefoil structure. TFFs are mainly expressed in the gastrointestinal tract where they play a critical role in the function of the mucosal barrier. TFF1 has been suggested as a neuropeptide, but not much is known about its expression and function in the central nervous system. We investigated the expression of TFF1 in the developing and adult rat midbrain. In the adult ventral mesencephalon, TFF1-immunoreactive (-ir) cells were predominantly found in the substantia nigra pars compacta (SNc), the ventral tegmental area (VTA) and in periaqueductal areas. While around 90% of the TFF1-ir cells in the SNc co-expressed tyrosine hydroxylase (TH), only a subpopulation of the TH-ir neurons expressed TFF1. Some TFF1-ir cells in the SNc co-expressed the calcium-binding proteins calbindin or calretinin and nearly all were NeuN-ir confirming a neuronal phenotype, which was supported by lack of co-localization with the astroglial marker glial fibrillary acidic protein (GFAP). Interestingly, at postnatal (P) day 7 and P14, a significantly higher proportion of TH-ir neurons in the SNc co-expressed TFF1 as compared to P21. In contrast, the proportion of TFF1-ir cells expressing TH remained unchanged during postnatal development. Furthermore, significantly more TH-ir neurons expressed TFF1 in the SNc, compared to the VTA at all four time-points investigated. Injection of the tracer fluorogold into the striatum of adult rats resulted in retrograde labeling of several TFF1 expressing cells in the SNc showing that a significant fraction of the TFF1-ir cells were projection neurons. This was also reflected by unilateral loss of TFF1-ir cells in SNc of 6-hydroxylase-lesioned hemiparkinsonian rats. In conclusion, we show for the first time that distinct subpopulations of midbrain dopaminergic neurons express TFF1, and that this expression pattern is altered in a rat model of Parkinson's disease.

Optical imaging of postsynaptic odor representation in the glomerular layer of the mouse olfactory bulb.

Olfactory glomeruli are the loci where the first odor-representation map emerges. The glomerular layer comprises exquisite local synaptic circuits for the processing of olfactory coding patterns immediately after their emergence. To understand how an odor map is transferred from afferent terminals to postsynaptic dendrites, it is essential to directly monitor the odor-evoked glomerular postsynaptic activity patterns. Here we report the use of a transgenic mouse expressing a Ca(2+)-sensitive green fluorescence protein (GCaMP2) under a Kv3.1 potassium-channel promoter. Immunostaining revealed that GCaMP2 was specifically expressed in mitral and tufted cells and a subpopulation of juxtaglomerular cells but not in olfactory nerve terminals. Both in vitro and in vivo imaging combined with glutamate receptor pharmacology confirmed that odor maps reported by GCaMP2 were of a postsynaptic origin. These mice thus provided an unprecedented opportunity to analyze the spatial activity pattern reflecting purely postsynaptic olfactory codes. The odor-evoked GCaMP2 signal had both focal and diffuse spatial components. The focalized hot spots corresponded to individually activated glomeruli. In GCaMP2-reported postsynaptic odor maps, different odorants activated distinct but overlapping sets of glomeruli. Increasing odor concentration increased both individual glomerular response amplitude and the total number of activated glomeruli. Furthermore, the GCaMP2 response displayed a fast time course that enabled us to analyze the temporal dynamics of odor maps over consecutive sniff cycles. In summary, with cell-specific targeting of a genetically encoded Ca(2+) indicator, we have successfully isolated and characterized an intermediate level of odor representation between olfactory nerve input and principal mitral/tufted cell output.

Glutamate and nitric oxide as regulatory transmitters in developing rabbit retina

Glutamate is the major excitatory neurotransmitter in the retina and serves as the synaptic messenger for the three classes of neurons which constitute the vertical pathway--the photoreceptors, bipolar cells and ganglion cells. In addition, the glutamate system has been localized morphologically, pharmacologically as well as molecularly during the first postnatal week of development before synaptogenesis occurs. The role which glutamate plays in the maturing visual system is complex but ranges from mediating developmental neurotoxicity to inducing neurite outgrowth.^ Nitric oxide/cGMP is a novel intercellular messenger which is thought to act in concert with the glutamate system in regulating a variety of cellular processes in the brain as well as retina, most notably neurotoxicity. Several developmental activities including programmed cell death, synapse elimination and synaptic reorganization are possible functions of cellular regulation modulated by nitric oxide as well as glutamate.^ The purpose of this thesis is to (1) biochemically characterize the endogenous pools of glutamate and determine what fraction exists extracellularly; (2) examine the morphological expression of NO producing cells in developing retina; (3) test the functional coupling of the NMDA subtype of glutamate receptor to the NO system by examining neurotoxicity which has roles in both the maturing and adult retina.^ Biochemical sampling of perfusates collected from the photoreceptor surface of ex vivo retina demonstrated that although the total pool of glutamate present at birth is relatively modest, a high percentage resides in extracellular pools. As a result, immature neurons without significant synaptic connections survive and develop in a highly glutamatergic environment which has been shown to be toxic in the adult retina.^ The interaction of the glutamate system with the NO system has been postulated to regulate neuronal survival. We therefore examined the developmental expression of the enzyme responsible for producing NO, nitric oxide synthase (NOS), using an antibody to the constitutive form of NOS found in the brain. The neurons thought to produce the majority of NO in the adult retina, a subpopulation of widefield amacrine cells, were not immunoreactive until the end of the second postnatal week. However, a unique developmental expression was observed in the ganglion cell layer and developing outer nuclear layer of the retina during the first postnatal week. We postulate NO producing neurons may not be present in a mature configuration therefore permitting neuronal survival in a highly glutamatergic microenvironment and allowing NO to play a development-specific role at this time.^ The next set of experiments constituted a functional test of the hypothesis that the absence of the prototypic NO producing cells in developing retina protects immature neurons against glutamate toxicity. An explant culture system developed in order to examine cellular responses of immature and adult neurons to glutamate toxicity showed that immature neurons were affected by NMDA but were less responsive to NMDA and NO mediated toxicity. In contrast, adult explants exhibited significant NMDA toxicity which was attenuated by NMDA antagonists, 2-amino-5-phosphonovaleric acid (APV), dextromethorphan (Dex) and N$\rm\sp{G}$-D-methyl arginine (metARG). These results indicated that pan-retinal neurotoxicity via the NMDA receptor and/or NO activation occurred in the adult retina but was not significant in the neonate. (Abstract shortened by UMI.) ^

Reelin immunoreactivity in dissociated cultures of the postnatal hippocampus

Reelin is an extracellular matrix glycoprotein expressed in different nerve cell populations in the developing, early postnatal and adult central nervous system. During histogenesis of the neocortex and hippocampus, reelin is present in Cajal-Retzius cells and other early neurons and contributes to correct layering of these regions. During early postnatal life, pioneer neurons disappear and reelin expression establishes in a subpopulation of cortical and hippocampal GABAergic interneurons, where it is maintained throughout adult life. We studied the developmental distribution pattern of reelin in dissociated cultures obtained from the early postnatal hippocampus to verify whether or not such a maturation phenomenon is maintained in vitro. Reelin is expressed both in Cajal-Retzius cells and multipolar and pyramidal neurons in younger cultures. The density of reelin-positive Cajal-Retzius cells dropped drastically by about 84% in 4-week-old cultures. Multipolar and pyramidal neurons containing reelin represented 12% of the total cell population in younger cultures and decreased by about 25% after 3 to 4 weeks of cultivation. Their density was significantly lower in cultures of the same age treated with glutamate receptor antagonists. These reelin-positive multipolar and pyramidal neurons were heterogeneous, including a larger amount of non-GABAergic, and 30-40% of GABAergic neurons. Cells double labeled for reelin and the GABA synthesizing enzyme glutamic acid decarboxylase represented about 4% of the total neuron population in culture and their density remained constant with age. It is thus possible that the decrease in the total reelin population may selectively be of importance to the larger non-GABAergic fraction of reelin cells. This study shows that reelin-expressing neurons are maintained in dissociated cultures of the neonatal hippocampus and their distribution and age-dependent changes in density resemble those of the early postnatal hippocampus in vivo.

Sono-Electro-Magnetic Therapy for Treating Chronic Pelvic Pain Syndrome in Men: A Randomized, Placebo-Controlled, Double-Blind Trial.

OBJECTIVE To assess the efficacy and safety of sono-electro-magnetic therapy compared to placebo in men with refractory CPPS. PATIENTS AND METHODS In a randomized, placebo-controlled, double-blind single center trial, we assessed the effect of sono-electro-magnetic therapy in men with treatment refractory CPPS. Sixty male patients were randomly assigned to treatment with either sono-electro-magnetic (n = 30) or placebo therapy (n = 30) for 12 weeks. The primary outcome was a change in the National Institutes of Health Chronic Prostatitis Symptom Index (NIH-CPSI) from baseline to 12 weeks. RESULTS The 12-week difference between sono-electro-magnetic and placebo therapy in changes of the NIH-CPSI total score was -3.1 points (95% CI -6.8 to 0.6, p = 0.11). In secondary comparisons of NIH-CPSI sub-scores, we found differences between groups most pronounced for the quality-of-life sub-score (difference at 12 weeks -1.6, 95% CI -2.8 to -0.4, p = 0.015). In stratified analyses, the benefit of sono-electro-magnetic therapy appeared more pronounced among patients who had a symptom duration of 12 months or less (difference in NIH-CPSI total score -8.3, 95% CI -14.5 to 2.6) than in patients with a longer symptom duration (-0.8, 95% CI -4.6 to 3.1; p for interaction = 0.023). CONCLUSIONS Sono-electro-magnetic therapy did not result in a significant improvement of symptoms in the overall cohort of treatment refractory CPPS patients compared to placebo treatment. Subgroup analysis indicates, however, that patients with a symptom-duration of 12 months or less may benefit from sono-electro-magnetic therapy, warranting larger randomized controlled trials in this subpopulation. TRIAL REGISTRATION ClinicalTrials.gov NCT00688506.

Regulation of hematopoietic and leukemic stem cells by the immune system.

Hematopoietic stem cells (HSCs) are rare, multipotent cells that generate via progenitor and precursor cells of all blood lineages. Similar to normal hematopoiesis, leukemia is also hierarchically organized and a subpopulation of leukemic cells, the leukemic stem cells (LSCs), is responsible for disease initiation and maintenance and gives rise to more differentiated malignant cells. Although genetically abnormal, LSCs share many characteristics with normal HSCs, including quiescence, multipotency and self-renewal. Normal HSCs reside in a specialized microenvironment in the bone marrow (BM), the so-called HSC niche that crucially regulates HSC survival and function. Many cell types including osteoblastic, perivascular, endothelial and mesenchymal cells contribute to the HSC niche. In addition, the BM functions as primary and secondary lymphoid organ and hosts various mature immune cell types, including T and B cells, dendritic cells and macrophages that contribute to the HSC niche. Signals derived from the HSC niche are necessary to regulate demand-adapted responses of HSCs and progenitor cells after BM stress or during infection. LSCs occupy similar niches and depend on signals from the BM microenvironment. However, in addition to the cell types that constitute the HSC niche during homeostasis, in leukemia the BM is infiltrated by activated leukemia-specific immune cells. Leukemic cells express different antigens that are able to activate CD4(+) and CD8(+) T cells. It is well documented that activated T cells can contribute to the control of leukemic cells and it was hoped that these cells may be able to target and eliminate the therapy-resistant LSCs. However, the actual interaction of leukemia-specific T cells with LSCs remains ill-defined. Paradoxically, many immune mechanisms that evolved to activate emergency hematopoiesis during infection may actually contribute to the expansion and differentiation of LSCs, promoting leukemia progression. In this review, we summarize mechanisms by which the immune system regulates HSCs and LSCs.

Cisplatin-resistant cells in malignant pleural mesothelioma cell lines show ALDH(high)CD44(+) phenotype and sphere-forming capacity

BACKGROUND Conventional chemotherapy in malignant pleural mesothelioma (MPM) has minimal impact on patient survival due to the supposed chemoresistance of cancer stem cells (CSCs). We sought to identify a sub-population of chemoresistant cells by using putative CSC markers, aldehyde dehydrogenase (ALDH) and CD44 in three MPM cell lines; H28, H2052 and Meso4. METHODS The Aldefluor assay was used to measure ALDH activity and sort ALDH(high) and ALDH(low) cells. Drug-resistance was evaluated by cell viability, anchorage-independent sphere formation, flow-cytometry and qRT-PCR analyses. RESULTS The ALDH(high) - and ALDH(low) -sorted fractions were able to demonstrate phenotypic heterogeneity and generate spheres, the latter being less efficient, and both showed an association with CD44. Cis- diamminedichloroplatinum (II) (cisplatin) treatment failed to reduce ALDH activity and conferred only a short-term inhibition of sphere generation in both ALDH(high) and ALDH(low) fractions of the three MPM cell lines. Induction of drug sensitivity by an ALDH inhibitor, diethylaminobenzaldehyde (DEAB) resulted in significant reductions in cell viability but not a complete elimination of the sphere-forming cells, suggestive of the presence of a drug-resistant subpopulation. At the transcript level, the cisplatin + DEAB-resistant cells showed upregulated mRNA expression levels for ALDH1A2, ALDH1A3 isozymes and CD44 indicating the involvement of these markers in conferring chemoresistance in both ALDH(high) and ALDH(low) fractions of the three MPM cell lines. CONCLUSIONS Our study shows that ALDH(high) CD44(+) cells are implicated in conveying tolerance to cisplatin in the three MPM cell lines. The combined use of CD44 and ALDH widens the window for identification and targeting of a drug-resistant population which may improve the current treatment modalities in mesothelioma.

Allele-specific polymerase chain reaction diagnostic test for the functional MDR1 polymorphism in dogs

The major multidrug transporter P-glycoprotein (Pgp) contributes to the barrier function of several tissues and organs, including the brain. In a subpopulation of Collies and seven further dog breeds, a 4 base pair deletion has been described in the Pgp-encoding MDR1 gene. This deletion results in the absence of a functional form of Pgp and loss of its protective function. Severe intoxication with the Pgp substrate ivermectin has been attributed to the genetically determined lack of Pgp. An allele-specific polymerase chain reaction (PCR)-based screening method has been developed to detect the mutant allele and to determine if a dog is homozygous or heterozygous for the mutation. Based on this validation, the allele-specific PCR proved to be a robust, reproducible and specific tool, allowing rapid determination of the MDR1 genotype of dogs of at risk breeds using blood samples or buccal swabs.

Using MDEFT MRI Sequences to Target the GPi in DBS Surgery.

OBJECTIVE Recent advances in different MRI sequences have enabled direct visualization and targeting of the Globus pallidus internus (GPi) for DBS surgery. Modified Driven Equilibrium Fourier Transform (MDEFT) MRI sequences provide high spatial resolution and an excellent contrast of the basal ganglia with low distortion. In this study, we investigate if MDEFT sequences yield accurate and reliable targeting of the GPi and compare direct targeting based on MDEFT sequences with atlas-based targeting. METHODS 13 consecutive patients considered for bilateral GPi-DBS for dystonia or PD were included in this study. Preoperative targeting of the GPi was performed visually based on MDEFT sequences as well as by using standard atlas coordinates. Postoperative CT imaging was performed to calculate the location of the implanted leads as well as the active electrode(s). The coordinates of both visual and atlas based targets were compared. The stereotactic coordinates of the lead and active electrode(s) were calculated and projected on the segmented GPi. RESULTS On MDEFT sequences the GPi was well demarcated in most patients. Compared to atlas-based planning the mean target coordinates were located significantly more posterior. Subgroup analysis showed a significant difference in the lateral coordinate between dystonia (LAT = 19.33 ± 0.90) and PD patients (LAT = 20.67 ± 1.69). Projected on the segmented preoperative GPi the active contacts of the DBS electrode in both dystonia and PD patients were located in the inferior and posterior part of the structure corresponding to the motor part of the GPi. CONCLUSIONS MDEFT MRI sequences provide high spatial resolution and an excellent contrast enabling precise identification and direct visual targeting of the GPi. Compared to atlas-based targeting, it resulted in a significantly different mean location of our target. Furthermore, we observed a significant variability of the target among the PD and dystonia subpopulation suggesting accurate targeting for each individual patient.

Expression of the hyaluronan-mediated motility receptor RHAMM in tumor budding cells identifies aggressive colorectal cancers.

Expression of the hyaluronan-mediated motility receptor (RHAMM, CD168) predicts adverse clinicopathological features and decreased survival for colorectal cancer (CRC) patients. Using full tissue sections, we investigated the expression of RHAMM in tumor budding cells of 103 primary CRCs to characterize the biological processes driving single-cell invasion and early metastatic dissemination. RHAMM expression in tumor buds was analyzed with clinicopathological data, molecular features and survival. Tumor budding cells at the invasive front of CRC expressed RHAMM in 68% of cases. Detection of RHAMM-positive tumor budding cells was significantly associated with poor survival outcome (P = .0312), independent of TNM stage and adjuvant therapy in multivariate analysis (P = .0201). RHAMM-positive tumor buds were associated with frequent lymphatic invasion (P = .0007), higher tumor grade (P = .0296), and nodal metastasis (P = .0364). Importantly, the prognostic impact of RHAMM expression in tumor buds was maintained independently of the number of tumor buds found in an individual case (P = .0246). No impact of KRAS/BRAF mutation, mismatch repair deficiency and CpG island methylation was observed. RHAMM expression identifies an aggressive subpopulation of tumor budding cells and is an independent adverse prognostic factor for CRC patients. These data support ongoing efforts to develop RHAMM as a target for precision therapy.

Propionibacterium acnes promotes Th17 and Th17/Th1 responses in acne patients

Propionibacterium acnes is a Gram-positive commensal bacterium thought to be involved in the pathogenesis of acne vulgaris. Although the ability of P. acnes in the initiation of pro-inflammatory responses is well documented, little is known about adaptive immune responses to this bacterium. The observation that infiltrating immune cells consist mainly of CD4(+) T cells in the perifollicular space of early acne lesions suggests that helper T cells may be involved in immune responses caused by the intra-follicular colonization of P. acnes. A recent report showing that P. acnes can induce IL-17 production by T cells suggests that acne might be a T helper type 17 (Th17)-mediated disease. In line with this, we show in this work that, in addition to IL-17A, both Th1 and Th17 effector cytokines, transcription factors, and chemokine receptors are strongly upregulated in acne lesions. Furthermore, we found that, in addition to Th17, P. acnes can promote mixed Th17/Th1 responses by inducing the concomitant secretion of IL-17A and IFN-γ from specific CD4(+) T cells in vitro. Finally, we show that both P. acnes-specific Th17 and Th17/Th1 cells can be found in the peripheral blood of patients suffering from acne and, at lower frequencies, in healthy individuals. We therefore identified P. acnes-responding Th17/Th1 cells as, to our knowledge, a previously unreported CD4(+) subpopulation involved in inflammatory acne.

High intratumoral levels of FoxP3+ lymphocytes are associated with better prognosis in primary resected esophageal adenocarcinomas.

Background: Tumor infiltrating T-lymphocytes (TILs) have been shown to play an important prognostic role in many carcinomas. The identification of prognostic relevant morphological or molecular factors is a major area of interest in the diagnostic process and for the treatment of highly aggressive esophageal adenocarcinoma. Studies about the impact of TILs in this tumor have not shown completely congruent results yet. We present a comprehensive study about the clinical and pathological impact of TIL in esophageal adenocarcinomas. Methods: A next generation tissue microarray (TMA) of 117 primary resected esophageal adenocarcinomas was analyzed for CD3+, CD8+ and FoxP3+ TIL using immunohistochemistry. The TMA contained three cores of the tumor center and the tumor periphery per each case. Slides were scanned with a high-resolution scanner (ScanScope CS; Aperio) and an image analysis software (Aperio Image Scope) was used to determine the TIL counts. The results were correlated with clinicopathological parameters. Results: CD3+, CD8+ and FoxP3+ TIL counts showed a significant correlation among each other (p<0.001 each, range: 0.27-0.77). TIL counts were categorized as high and low levels, according to the median. Tumors with high FoxP3+ intratumoral lymphocyte counts were more frequently of lower pT category (p<0.001) and without lymph node metastasis (p=0.04). High levels of FoxP3+ lymphocytes in the tumor center and the periphery were also associated with better prognosis (p<0.001 and p=0.041, respectively) in univariate analysis. A similar prognostic impact was seen for high levels of CD3+ and CD8+ TIL in the tumor center, but not in the periphery (p=0.047 and p=0.011, respectively). In multivariate analysis high central FoxP3+TIL levels were an independent prognostic factor (HR=0.4; p=0.023) which was similar to a combination score of CD3+/CD8+/FoxP3+ TIL (HR=0.54; p=0.027) or CD8+/Foxp3+ TIL (HR=0.052; p=0.020) and superior to pT- and pN category (p>0.05 each). Conclusion: This study demonstrates a significant beneficial prognostic impact of high TIL counts in the tumor center of esophageal adenocarcinomas, in particular with regards to the subpopulation of FoxP3+ and CD8+ T-regulatory cells. The determination of intratumoral lymphocytic counts and application of TIL scores can improve prognostic accuracy of pathologic reports of these tumors and may be helpful for better risk stratification of esophageal adenocarcinoma patients.

Pan-epicardial lineage tracing reveals that epicardium derived cells give rise to myofibroblasts and perivascular cells during zebrafish heart regeneration.

Myocardial infarction (MI) leads to a severe loss of cardiomyocytes, which in mammals are replaced by scar tissue. Epicardial derived cells (EPDCs) have been reported to differentiate into cardiomyocytes during development, and proposed to have cardiomyogenic potential in the adult heart. However, mouse MI models reveal little if any contribution of EPDCs to myocardium. In contrast to adult mammals, teleosts possess a high myocardial regenerative capacity. To test if this advantage relates to the properties of their epicardium, we studied the fate of EPDCs in cryoinjured zebrafish hearts. To avoid the limitations of genetic labelling, which might trace only a subpopulation of EPDCs, we used cell transplantation to track all EPDCs during regeneration. EPDCs migrated to the injured myocardium, where they differentiated into myofibroblasts and perivascular fibroblasts. However, we did not detect any differentiation of EPDCs nor any other non-cardiomyocyte population into cardiomyocytes, even in a context of impaired cardiomyocyte proliferation. Our results support a model in which the epicardium promotes myocardial regeneration by forming a cellular scaffold, and suggests that it might induce cardiomyocyte proliferation and contribute to neoangiogenesis in a paracrine manner.