In Vivo Efficacy of Neutrophil-Mediated Bone Regeneration Using a Rabbit Calvarial Defect Model.

Reconstruction of bone due to surgical removal or disease-related bony defects is a clinical challenge. It is known that the immune system exerts positive immunomodulatory effects on tissue repair and regeneration. In this study, we evaluated the in vivo efficacy of autologous neutrophils on bone regeneration using a rabbit calvarial defect model. Methods: Twelve rabbits, each with two surgically created calvarial bone defects (10 mm diameter), were randomly divided into two groups; (i) single application of neutrophils (SA-NP) vs. SA-NP control, and (ii) repetitive application of neutrophils (RA-NP) vs. RA-NP control. The animals were euthanized at 4 and 8 weeks post-operatively and the treatment outcomes were evaluated by micro-computed tomography, histology, and histomorphometric analyses. Results: The micro-CT analysis showed a significantly higher bone volume fraction (bone volume/total volume) in the neutrophil-treated groups, i.e., median interquartile range (IQR) SA-NP (18) and RA-NP (24), compared with the untreated controls, i.e., SA-NP (7) and RA-NP (14) at 4 weeks (p < 0.05). Similarly, new bone area fraction (bone area/total area) was significantly higher in neutrophil-treated groups at 4 weeks (p < 0.05). Both SA-NP and RA-NP had a considerably higher bone volume and bone area at 8 weeks, although the difference was not statistically significant. In addition, immunohistochemical analysis at 8 weeks revealed a higher expression of osteocalcin in both SA-NP and RA-NP groups. Conclusions: The present study provides first hand evidence that autologous neutrophils may have a positive effect on promoting new bone formation. Future studies should be performed with a larger sample size in non-human primate models. If proven feasible, this new promising strategy could bring clinical benefits for bone defects to the field of oral and maxillofacial surgery.

Non-neuronal acetylcholine involved in reproduction in mammals and honeybees.

Bacteria and archaea synthesize acetylcholine (ACh). Thus, it can be postulated that ACh was created by nature roughly three billion years ago. Therefore, the wide expression of ACh in nature (i.e., in bacteria, archaea, unicellular organisms, plants, fungi, non-vertebrates and vertebrates and in the abundance of non-neuronal cells of mammals) is not surprising. The term non-neuronal ACh and non-neuronal cholinergic system have been introduced to describe the auto- and paracrine, that is, local regulatory actions of ACh in cells not innervated by neuronal cholinergic fibers and to communicate among themselves. In this way non-neuronal ACh binds to the nicotinic or muscarinic receptors expressed on these local and migrating cells and modulates basic cells functions such as proliferation, differentiation, migration and the transport of ions and water. The present article is focused to the effects of non-neuronal ACh linked to reproduction; data on the expression and function of the non-neuronal cholinergic system in the following topics are summarized: (i) Sperm, granulosa cells, oocytes; (ii) Auxiliary systems (ovary, oviduct, placenta); (iii) Embryonic stem cells as first step for reproduction of a new individual after fertilization; (iv) Larval food as an example of reproduction in insects (honeybees) and adverse effects of the neonicotinoids, a class of world-wide applied insecticides. The review article will show that non-neuronal ACh is substantially involved in the regulation of reproduction in mammals and also non-mammals like insects (honeybees). There is a need to learn more about this biological role of ACh. In particular, we have to consider that insecticides like the neonicotinoids, but also carbamates and organophosphorus pesticides, interfere with the non-neuronal cholinergic system thus compromising for example the breeding of honeybees. But it is possible that other species may also be adversely affected as well, a mechanism which may contribute to the observed decline in biodiversity. This is an article for the special issue XVth International Symposium on Cholinergic Mechanisms.

Macrophage type modulates osteogenic differentiation of adipose tissue MSCs.

Since the reconstruction of large bone defects remains a challenge, knowledge about the biology of bone healing is desirable to develop novel strategies for improving the treatment of bone defects. In osteoimmunology, macrophages are the central component in the early stage of physiological response after bone injury and bone remodeling in the late stage. During this process, a switch of macrophage phenotype from pro-inflammatory (M1) to anti-inflammatory (M2) is observed. An appealing option for bone regeneration would be to exploit this regulatory role for the benefit of osteogenic differentiation of osteoprogenitor cells (e.g., mesenchymal stem cells; MSCs) and to eventually utilize this knowledge to improve the therapeutic outcome of bone regenerative treatment. In view of this, we focused on the in vitro interaction of different macrophage subtypes with adipose tissue MSCs to monitor the behavior (i.e. proliferation, differentiation and mineralization) of the latter in dedicated co-culture models. Our data show that co-culture of MSCs with M2 macrophages, but not with M1 macrophages or M0 macrophages, results in significantly increased MSC mineralization caused by soluble factors. Specifically, M2 macrophages promoted the proliferation and osteogenic differentiation of MSCs, while M0 and M1 macrophages solely stimulated the osteogenic differentiation of MSCs in the early and middle stages during co-culture. Secretion of the soluble factors oncostatin M (OSM) and bone morphogenetic protein 2 (BMP-2) by macrophages showed correlation with MSC gene expression levels for OSM-receptor and BMP-2, suggesting the involvement of both signaling pathways in the osteogenic differentiation of MSCs.

Influenza virus damages the alveolar barrier by disrupting epithelial cell tight junctions.

A major cause of respiratory failure during influenza A virus (IAV) infection is damage to the epithelial-endothelial barrier of the pulmonary alveolus. Damage to this barrier results in flooding of the alveolar lumen with proteinaceous oedema fluid, erythrocytes and inflammatory cells. To date, the exact roles of pulmonary epithelial and endothelial cells in this process remain unclear.Here, we used an in vitro co-culture model to understand how IAV damages the pulmonary epithelial-endothelial barrier. Human epithelial cells were seeded on the upper half of a transwell membrane while human endothelial cells were seeded on the lower half. These cells were then grown in co-culture and IAV was added to the upper chamber.We showed that the addition of IAV (H1N1 and H5N1 subtypes) resulted in significant barrier damage. Interestingly, we found that, while endothelial cells mounted a pro-inflammatory/pro-coagulant response to a viral infection in the adjacent epithelial cells, damage to the alveolar epithelial-endothelial barrier occurred independently of endothelial cells. Rather, barrier damage was associated with disruption of tight junctions amongst epithelial cells, and specifically with loss of tight junction protein claudin-4.Taken together, these data suggest that maintaining epithelial cell integrity is key in reducing pulmonary oedema during IAV infection.

Histopathological features predict metastatic potential in locally advanced colon carcinomas.

BACKGROUND: Metastatic dissemination can exist before a pathologically and clinically detectable manifestation. The structural heterogeneity of colon cancer (CC) in histological sections with respect to the morphology of tumor aggressiveness and composition of the tumor microenvironment raises the question of whether the microscopical tumor architecture enables a discrimination of groups with different metastatic potential. This would result in an assessment of the prognosis and provision of an ancillary tool for the therapeutic management after surgery, beside the estimation of the local tumor extent. METHODS: In order to identify predictive biomarkers for metastasis of locally advanced CC, which can easily be integrated into the pathologist's daily routine diagnostic activity, we determined tumor budding, peritumoral inflammation, extent of desmoplasia and necrosis, density of macro- and microvascular blood vessels and functional state of lymphatics in the tumor center, invasive margin and tumor-free surrounding tissue in 86 non-metastatic, lymphogenous-metastatic and haematogenous-metastatic, subserosa-invasive CC. RESULTS: Features influencing nodal metastasis in the univariate analysis included high tumor budding (p = 0.004), high large vessel density in the subserosa (p = 0.043), abundant desmoplasia (p = 0.049), non-finger-like desmoplastic pattern (p = 0.051) and absent lymphocellular intratumoral inflammation (p = 0.084). In the multivariate analysis, with the exception of large vessel density, these pathomorphological features were independent risk factors for lymphogenous metastasis (p = 0.023, p = 0.017, p = 0.037, p = 0.012, respectively) with a good discrimination ability (AUC of 0.853). Features associated with distant metastasis in the univariate analysis included high tumor budding (p = 0.002), low intratumoral small vessel density (p = 0.013), absent lymphocellular intratumoral inflammation (p = 0.048) and abundant necrosis (p = 0.073). In the multivariate analysis only tumor budding was an independent predictor for haematogenous metastasis (p = 0.007) with a good discrimination ability (AUC of 0.829). CONCLUSIONS: Thus, mainly tumor budding but also the described structural characteristics of the peritumoral tissue appears to reflect the metastatic potential of locally advanced CC and therefore should be stated in pathological reports.

Cell type- and tumor zone-specific expression of pVEGFR-1 and its ligands influence colon cancer metastasis.

BACKGROUND: Detailed knowledge of the essential pro-angiogenic biomolecules, the vascular endothelial growth factor (VEGF) family and its receptors, in the characteristically heterogeneous tumor tissue is a pre-requisite for an effective personalized target therapy. The effects of VEGF receptors after ligand binding are mediated through receptor tyrosine autophosphorylation. We determined the relevance of the VEGFR-1 activating pathway for colon cancer (CC) metastasis. METHODS: The expression profiles of VEGFR-1, phosphorylated (activated) VEGFR-1 (pVEGFR-1(Tyr1048), pVEGFR-1(Tyr1213) and pVEGFR-1(Tyr1333)) and the VEGFR-1 ligands (VEGF, PlGF and VEGF-B) were investigated using immunohistochemistry in different tumor compartments (intratumoral - invasive front - extratumoral), cell types (tumor cells - macro- (large and small vessels) and the microvasculature (capillaries) - inflammatory cells) in human sporadic non-metastatic, lymphogenous metastatic and haematogenous metastatic CC. RESULTS: VEGF and PlGF produced by tumor cells have an autocrine affinity for their receptor VEGFR-1. Subsequent PlGF-mediated receptor activation by autophosphorylation at Tyr1048 and Tyr1213 is a potential signaling pathway, which in turn seems to protect against distant metastasis and, in regions of tumor budding, additionally against lymph node metastasis. This autocrine link could be supported by possible formation of PlGF-VEGF heterodimers and PlGF-PlGF homodimers, which are known to have anti-metastatic properties. In contrast, in order to enhance their potential for distant metastasis tumor cells produce paracrine-acting VEGF-B. VEGFR-1 activation in tumor-associated macrovasculature but not capillaries appears to affect metastatic ability. Paracrine-mediated receptor autophosphorylation at Tyr1048 and Tyr1213 in small vessels located intratumorally and along the invasive front appears to be inversely correlated with metastasis, especially distant metastasis. Additionally, macrovessels are able to produce VEGFR-1 ligands, which influence the metastatic potential. Paracrine-acting VEGF-B production by intratumorally located small vessels and autocrine-acting PlGF production by extratumorally located small vessels seem to be associated with the non-metastatic phenotype. In contrast, VEGF-B-expressing extratumoral large and small vessels correlate with distant metastasis. Lymphocyte-associated VEGFR-1 expression in the invasive front without accompanying autophosphorylation could prevent against distant metastasis possibly by acting as a decoy and scavenger receptor. CONCLUSION: VEGFR-1 and its ligands participate in vascular, tumor cell-mediated and immuno-inflammatory processes in a complex biomolecule-dependent and tumor zone-specific manner and hence could influence metastatic behavior in CC.

Activation of muscarinic receptors by non-neuronal acetylcholine.

The biological role of acetylcholine and the cholinergic system is revisited based particularly on scientific research early and late in the last century. On the one hand, acetylcholine represents the classical neurotransmitter, whereas on the other hand, acetylcholine and the pivotal components of the cholinergic system (high-affinity choline uptake, choline acetyltransferase and its end product acetylcholine, muscarinic and nicotinic receptors and esterase) are expressed by more or less all mammalian cells, i.e. by the majority of cells not innervated by neurons at all. Moreover, it has been demonstrated that acetylcholine and "cholinergic receptors" are expressed in non-neuronal organisms such as plants and protists. Acetylcholine is even synthesized by bacteria and algae representing an extremely old signalling molecule on the evolutionary timescale. The following article summarizes examples, in which non-neuronal acetylcholine is released from primitive organisms as well as from mammalian non-neuronal cells and binds to muscarinic receptors to modulate/regulate phenotypic cell functions via auto-/paracrine pathways. The examples demonstrate that non-neuronal acetylcholine and the non-neuronal cholinergic system are vital for various types of cells such as epithelial, endothelial and immune cells.

Covalent linkage of sulfated hyaluronan to the collagen scaffold Mucograft® enhances scaffold stability and reduces proinflammatory macrophage activation in vivo.

Sulfated glycosaminoglycans (sGAG) show interaction with biological mediator proteins. Although collagen-based biomaterials are widely used in clinics, their combination with high-sulfated hyaluronan (sHA3) is unexplored. This study aims to functionalize a collagen-based scaffold (Mucograft®) with sHA3 via electrostatic (sHA3/PBS) or covalent binding to collagen fibrils (sHA3+EDC/NHS). Crosslinking without sHA3 was used as a control (EDC/NHS Ctrl). The properties of the sHA3-functionalized materials were characterized. In vitro growth factor and cytokine release after culturing with liquid platelet-rich fibrin was performed by means of ELISA. The cellular reaction to the biomaterials was analyzed in a subcutaneous rat model. The study revealed that covalent linking of sHA3 to collagen allowed only a marginal release of sHA3 over 28 days in contrast to electrostatically bound sHA3. sHA3+EDC/NHS scaffolds showed reduced vascular endothelial growth factor (VEGF), transforming growth factor beta 1 (TGF-ß1) and enhanced interleukin-8 (IL-8) and epithelial growth factor (EGF) release in vitro compared to the other scaffolds. Both sHA3/PBS and EDC/NHS Ctrl scaffolds showed a high proinflammatory reaction (M1: CD-68+/CCR7+) and induced multinucleated giant cell (MNGC) formation in vivo. Only sHA3+EDC/NHS scaffolds reduced the proinflammatory macrophage M1 response and did not induce MNGC formation during the 30 days. SHA3+EDC/NHS scaffolds had a stable structure in vivo and showed sufficient integration into the implantation region after 30 days, whereas EDC/NHS Ctrl scaffolds underwent marked disintegration and lost their initial structure. In summary, functionalized collagen (sHA3+EDC/NHS) modulates the inflammatory response and is a promising biomaterial as a stable scaffold for full-thickness skin regeneration in the future.

Multinucleated Giant Cells Induced by a Silk Fibroin Construct Express Proinflammatory Agents: An Immunohistological Study.

Multinucleated giant cells (MNGCs) are frequently observed in the implantation areas of different biomaterials. The main aim of the present study was to analyze the long-term polarization pattern of the pro- and anti-inflammatory phenotypes of macrophages and MNGCs for 180 days to better understand their role in the success or failure of biomaterials. For this purpose, silk fibroin (SF) was implanted in a subcutaneous implantation model of Wistar rats as a model for biomaterial-induced MNGCs. A sham operation was used as a control for physiological wound healing. The expression of different inflammatory markers (proinflammatory M1: CCR-7, iNos; anti-inflammatory M2: CD-206, CD-163) and tartrate-resistant acid phosphatase (TRAP) and CD-68 were identified using immunohistochemical staining. The results showed significantly higher numbers of macrophages and MNGCs within the implantation bed of SF-expressed M1 markers, compared to M2 markers. Interestingly, the expression of proinflammatory markers was sustained over the long observation period of 180 days. By contrast, the control group showed a peak of M1 macrophages only on day 3. Thereafter, the inflammatory pattern shifted to M2 macrophages. No MNGCs were observed in the control group. To the best of our knowledge, this is study is the first to outline the persistence of pro-inflammatory MNGCs within the implantation bed of SF and to describe their long-term kinetics over 180 days. Clinically, these results are highly relevant to understand the role of biomaterial-induced MNGCs in the long term. These findings suggest that tailored physicochemical properties may be a key to avoiding extensive inflammatory reactions and achieving clinical success. Therefore, further research is needed to elucidate the correlation between proinflammatory MNGCs and the physicochemical characteristics of the implanted biomaterial.

Improved detection of sentinel lymph node metastases allows reliable intraoperative identification of patients with extended axillary lymph node involvement in early breast cancer.

BACKGROUND: An improved procedure that allows accurate detection of negative sentinel lymph node (SLN) and of SLN macrometastases during surgery would be highly desirable in order to protect patients from further surgery and to avoid unnecessary costs. We evaluated the accuracy of an intraoperative procedure that combines touch imprint cytology (TIC) and subsequent frozen section (FS) analysis. 2276 SLNs from 1072 patients with clinical node-negative early breast cancer were evaluated during surgery using TIC. Only cytologically-positive SLN were subsequently analysed with a single FS, preserving cytologically-negative SLN for the final postoperative histological diagnosis. Sensitivity, specificity and the accuracy of this approach were analysed by comparing the results from intra- and postoperative SLN and axillary node evaluation. This intraoperative method displayed 100% specificity for SLN metastases and was significantly more sensitive for prognostically relevant macrometastases (85%) than for micrometastases (10%). Sensitivity was highest for patients with two or more positive LNs (96%) than for those with only one (72%). 98% of the patients with final pN2a-pN3a were already identified during surgery. Patients who received primary axillary lymph node dissection had significantly more frequent metastases in further LNs (44.6%). Sensitivity was highest for patients with luminal-B, HER2+ and triple negative breast cancer and for any subtype if Ki-67 > 40%. TIC and subsequent FS of cytologically-positive SLNs is highly reliable for detection of SLN macrometastases, and allows accurate identification of patients with a high risk of extended axillary involvement during surgery, as well as accurate histological diagnosis of negative SLN.

Thermal treatment at 500 °C significantly reduces the reaction to irregular tricalcium phosphate granules as foreign bodies: An in vivo study.

Evaporation of phosphate species during thermal treatment (> 400 °C) of calcium phosphates leads to the formation of an alkaline layer on their surface. The aim of this study was to evaluate the hypothesis that the biological response of thermally treated calcium phosphates is modified by the presence of such an alkaline layer on their surface. For this purpose, 0.125-0.180 mm α- and ß-tricalcium phosphate (TCP) granules were obtained by crushing and size classification, with some being subjected to thermal treatment at 500 °C. The four types of granules (α-TCP, ß-TCP, α-TCP-500 °C, and ß-TCP-500 °C) were implanted subcutaneously and orthotopically in rats. Sham operations served as control. Subcutaneously, α-TCP and ß-TCP induced significantly more multinucleated giant cells (MNGCs) than calcined granules. Most of the induced MNGCs were TRAP-negative, CD-68 positive and cathepsin K-negative, reflecting a typical indication of a reaction with a foreign body. The vessel density was significantly higher in the α-TCP and ß-TCP groups than it was in the α-TCP-500 °C and ß-TCP-500 °C groups. In the femur model, ß-TCP-500 °C induced significantly more new bone formation than that induced by ß-TCP. The granule size was also significantly larger in the ß-TCP-500 °C group, making it more resistant to degradation than ß-TCP. The MNGC density was higher in the α-TCP and ß-TCP groups than in the α-TCP-500 °C and ß-TCP-500 °C groups, including cathepsin-positive, CD-68 positive, TRAP-positive and TRAP-negative MNGCs. In conclusion, this study confirms that the biological response of calcium phosphates was affected by the presence of an alkaline layer on their surface. Thermally-treated α-TCP and ß-TCP granules produced significantly fewer MNGCs and were significantly less degraded than non-thermally-treated α-TCP and ß-TCP granules. Thermally treating α-TCP and ß-TCP granules shifts the reaction from a foreign body reaction towards a physiological reaction by downregulating the number of induced MNGCs and enhancing degradation resistance.

Side-specific effects by cadmium exposure: apical and basolateral treatment in a coculture model of the blood-air barrier.

Cadmium (Cd(2+)) is a widespread environmental pollutant, which is associated with a wide variety of cytotoxic and metabolic effects. Recent studies showed that intoxication with the heavy metal most importantly targets the integrity of the epithelial barrier. In our study, the lung epithelial cell line, NCI H441, was cultured with the endothelial cell line, ISO-HAS-1, as a bilayer on a 24-well HTS-Transwell filter plate. This coculture model was exposed to various concentrations of CdCl(2). The transepithelial electrical resistance decreased on the apical side only after treatment with high Cd(2+) concentrations after 48 h. By contrast, a breakdown of TER to less than 5% of baseline could be observed much earlier (after 24 h) when Cd(2+) was administered from the basal side. Observations of cell layer fragmentation and widening of intercellular spaces confirmed the barrier breakdown only for the basolaterally treated samples. Furthermore, the cytotoxicity and release of proinflammatory markers was enhanced if samples were exposed to Cd(2+) from the basal side compared to treatment from the apical side. Moreover, we could demonstrate that a high concentration of Ca(2+) could prevent the barrier-disrupting effect of Cd(2+). In conclusion, the exposure of Cd(2+) to cocultures of lung cells caused a decrease in TER, major morphological changes, a reduction of cell viability and an increase of cytokine release, but the effects markedly differed between the two modes of exposure. Therefore, our results suggest that intact epithelial TJs may play a major role in protecting the air-blood barrier from inhaled Cd(2+).

Cholinergic signaling controls immune functions and promotes homeostasis.

Acetylcholine (ACh) was created by nature as one of the first signaling molecules, expressed already in procaryotes. Based on the positively charged nitrogen, ACh could initially mediate signaling in the absence of receptors. When evolution established more and more complex organisms the new emerging organs systems, like the smooth and skeletal muscle systems, energy-generating systems, sexual reproductive system, immune system and the nervous system have further optimized the cholinergic signaling machinery. Thus, it is not surprising that ACh and the cholinergic system are expressed in the vast majority of cells. Consequently, multiple common interfaces exist, for example, between the nervous and the immune system. Research of the last 20 years has unmasked these multiple regulating mechanisms mediated by cholinergic signaling and thus, the biological role of ACh has been revised. The present article summarizes new findings and describes the role of both non-neuronal and neuronal ACh in protecting the organism from external and internal health threats, in providing energy for the whole organism and for the individual cell, controling immune functions to prevent inflammatory dysbalance, and finally, the involvement in critical brain functions, such as learning and memory. All these capacities of ACh enable the organism to attain and maintain homeostasis under changing external conditions. However, the existence of identical interfaces between all these different organ systems complicates the research for new therapeutic interventions, making it essential that every effort should be undertaken to find out more specific targets to modulate cholinergic signaling in different diseases.

Co-culture Model for Cutaneous Wound Healing to Assess a Porous Fiber-Based Drug Delivery System.

In vitro tissue-engineered cell culture models are an essential instrument to investigate physiological and pathophysiological wound healing mechanisms and to evaluate new beneficial wound dressing materials and therapeutics to identify possible drug targets and to improve regeneration processes in nonhealing and chronic wounds. In this study, the authors established an in vitro model for cutaneous wound healing, based on primary human dermal microvascular endothelial cells (HDMEC) and primary human dermal fibroblasts (HDF) to study wound healing-associated processes. Co-cultivation of HDMEC and HDF results in the formation of microvessel-like structures in long-term co-cultures. The proposed in vitro co-culture model can be easily modified by adding macrophages to simulate the process of inflammation, thus allowing in vitro investigation of pathophysiological wound healing processes present in nonhealing wounds. Furthermore, the beneficial in vitro wound healing model was used to evaluate a porous fiber-based drug delivery dressing material consisting of melt-spun porous fibers that were filled with a hydrogel carrier (gellan gum) containing vascular endothelial growth factor (VEGF). Angiogenic capability was chosen as functional parameter for improved wound healing, and release of deposited VEGF from the dressing material was evaluated up to 7 days of cultivation. The experiments demonstrated that the porous fiber-based drug delivery dressing material for dermal wound healing with incorporated VEGF strongly enhances the process of angiogenesis in the in vitro co-culture model through a release of VEGF over 7 days of cultivation. In conclusion, tissue-engineered human skin equivalents could contribute significantly to the understanding and improvement of drug releasing dressing materials in the context of treating chronic wounds.

Comparative study of human colonic tumor-derived endothelial cells (HCTEC) and normal colonic microvascular endothelial cells (HCMEC): Hypoxia-induced sVEGFR-1 and sVEGFR-2 levels.

Colorectal carcinoma growth and progression is dependent on the vasculature of the tumor microenvironment. Tumor-derived endothelial cells differ functionally from their normal counterpart. For this reason we isolated microvascular endothelial cells from human colon cancer tissue (HCTEC) and compared them with endothelial cells from normal colonic tissue (HCMEC) of the same donor. Since hypoxia is a universal hallmark of carcinomas, we examined its effects on HCTEC of five patients in comparison with the corresponding HCMEC, with respect to the secretion of the soluble form of the two important vascular endothelial growth factor (VEGF) receptors, VEGFR-1 and -2. After dissociation by dispase/collagenase of central non-necrotic tumor areas obtained from colon carcinomas, HCTEC were isolated using CD31-coated magnetic beads and cultivated as monolayers. Subsequent characterization studies demonstrated the endothelial phenotype, including VEGFR-1 and -2 mRNA and protein expression as well as E-selectin expression, up-regulated after LPS, TNFalpha and IL-1beta stimulation. sVEGFR expression analyses were performed using ELISA. In comparison with HCMEC markedly lower sVEGFR-1 protein concentrations were found in HCTEC. These low sVEGFR-1 levels remain unchanged under hypoxia. In contrast, sVEGFR-2 was significantly decreased in both HCMEC and HCTEC under hypoxic conditions (p

Hypoxia-induced reduction of sVEGFR-2 levels in human colonic microvascular endothelial cells in vitro: Comparative study with HUVEC.

The functionality of large-vessel endothelial cells, such as human umbilical vein endothelial cells (HUVEC), may differ significantly from that in the microvasculature. We established a method for the isolation of human colonic microvascular endothelial cells (HCMEC). Since colonic diseases are often accompanied by hypoxia we examined its effects on HCMEC of five individuals in comparison with HUVEC, with respect to the secretion of the soluble form of the two important vascular endothelial growth factor (VEGF) receptors, VEGFR-1 and 2. After dissociation by dispase/collagenase of mucosal and submucosal tissue obtained from normal adult colon, HCMEC were isolated using CD31-coated magnetic beads and cultivated as monolayers. Subsequent characterization studies demonstrated the endothelial phenotype, including VEGFR-1 and 2 mRNA and protein expression. sVEGFR expression analyses were performed using ELISA. Under hypoxic conditions significantly enhanced levels of sVEGFR-1 on HUVEC were observed (p<0.001), while in HCMEC there was a markedly variable reaction to hypoxia, with cases of enhanced, unchanged and reduced expression. sVEGFR-2 was significantly decreased in HCMEC under hypoxia (p<0.001). In contrast, the responses of sVEGFR-2 levels to hypoxia in HUVEC were variable, that is, either unchanged or up-regulated. The different secretion profiles of sVEGFR-1 and 2 between HUVEC and HCMEC under normoxia and hypoxia underline the importance of using a functionally adequate and relevant microvasculature for in vitro studies of colonic diseases. The homogeneously reduced sVEGFR-2 levels in hypoxic HCMEC provide evidence for a novel microvascular endothelium-specific biomarker in hypoxia-response processes.

Biomimetic strategy towards gelatin coatings on PET. Effect of protocol on coating stability and cell-interactive properties.

Gelatin-modified poly(ethylene terephthalate) (PET) surfaces have been previously realized via an intermediate dopamine coating procedure that resulted in surfaces with superior haemocompatibility compared to unfunctionalized PET. The present study addresses the biocompatibility assessment of these coated PET surfaces. In this context, the stability of the gelatin coating upon exposure to physiological conditions and its cell-interactive properties were investigated. The proposed gelatin-dopamine-PET surfaces showed an increased protein coating stability up to 24 days and promoted the attachment and spreading of both endothelial cells (ECs) and smooth muscle cells (SMCs). In parallel, physisorbed gelatin coatings exhibited similar cell-interactive properties, albeit temporarily, as the coating delaminated within 1 week after cell seeding. Furthermore, no or only minimal immunogenic or inflammatory responses were observed during in vitro cytotoxicity and endotoxicity assessment for all gelatin-modified PET surfaces evaluated. Overall, the combined enhanced biocompatibility reported herein together with the previously proven haemocompatibility show the potential of the gelatin-dopamine-PET surfaces to function as vascular graft candidates.

Subendothelial infiltration of neutrophil granulocytes and liberation of matrix-destabilizing enzymes in an experimental model of human neo-intima.

It was the objective of this study to examine the role of human neutrophil granulocytes (PMN) in an in-vitro model of human neo-intima developed for the study of atherosclerosis. Human granulocytes were subjected to a co-culture model of human endothelial and smooth muscle cells. Subendothelial lipid accumulation was achieved by addition of native LDL to the culture medium. Tissue samples were analyzed by immunohistochemistry and scanning/transmission electron microscopy, and culture supernatants were examined for the presence of interleukin-8 (IL-8), MCP-1, GRO-alpha, elastase and matrixmetalloproteinase-8 (MMP-8). Following addition of 2 mg/ml LDL, adherence, transmigration and infiltration depth of PMN was increased significantly when compared to controls. LDL challenging was paralleled by a time- and dose-dependent secretion of IL-8 from intimal smooth muscle cells. PMN infiltration was mediated by the IL-8-signalling pathway and accompanied by release of elastase and MMP-8 into the supernatant and induction of endothelial cell apoptosis. In conclusion, LDL-induced secretion of IL-8 by intimal smooth muscle cells provides a potential mechanism of PMN-recruitment into culprit lesions. The concomitant release of potent matrix-degrading enzymes and the induction of EC apoptosis may have implications for plaque destabilization and cardiovascular events.

Hypoxia-induced epithelial VEGF-C/VEGFR-3 upregulation in carcinoma cell lines.

Adaptation to hypoxia, a universal hallmark of carcinomas, is a critical step for tumor cell survival and growth. One of the principal regulators of hypoxia-responsive pathways is the transcription factor hypoxia-inducible factor-1 alpha (HIF-1 alpha). Currently, it is known that tumoral production of members of the vascular endothelial growth factor (VEGF)-family (VEGFs) may promote tumor growth and progression by acting on carcinoma cells that express the cognate receptors (VEGFRs). However, the influence of hypoxia in the formation of such a tumoral VEGF/VEGFR loop is not completely understood. In the present study we examined the potential existence of a HIF-1 alpha/VEGF/VEGFR autocrine loop on commonly occurring carcinomas. The experiments were performed on five colorectal carcinoma cell lines, one breast (MCF7) and one lung (A549) adenocarcinoma cell line under normoxic and oxygen stress conditions using HIF-1 alpha-EIA, VEGFs-ELISA as well as RT-PCR and immunofluorescence for VEGFRs. HIF-1 alpha overexpression was found already after 2 h of exposure to hypoxia in all above mentioned cell lines, thus documenting that activation of the transcription factor HIF-1 alpha is an early cellular event. Under hypoxic conditions a significant upregulation and activation of HIF-1 alpha accompanied by an increased production of VEGF in MCF7 and A549 was observed. The well-differentiated colorectal carcinoma cell lines were 'hypoxia-resistant' showing unchanged levels of HIF-1 alpha and VEGF under hypoxia. None of the cell lines used in this study expressed the VEGF receptors VEGFR-1 and VEGFR-2 under normoxia and hypoxia. Additionally, all colorectal carcinoma cell lines were negative for VEGFR-3 transcripts in both conditions. However, VEGFR-3 mRNA and protein were expressed and under hypoxia overexpressed in MCF7 and A549. Hypoxic cultures of both cell lines secreted in elevated levels the VEGFR-3 ligand VEGF-C but not VEGF-D. Our findings suggest that under hypoxic conditions an autocrine loop between VEGF-C/VEGFR-3 and HIF-1 alpha is possible in breast carcinoma and lung carcinoma but not in colorectal carcinoma cell lines.

Hypoxia-inducible factor 1 alpha expression increases during colorectal carcinogenesis and tumor progression.

BACKGROUND: Hypoxia-inducible factor 1 alpha (HIF-1alpha) is involved in processes promoting carcinogenesis of many tumors. However, its role in the development of colorectal cancer is unknown. To investigate the significance of HIF-1alpha during colorectal carcinogenesis and progression we examined its expression in precursor lesions constituting the conventional and serrated pathways, as well as in non-metastatic and metastatic adenocarcinomas. METHODS: Immunohistochemistry and Western blot is used to analyse HIF-1alpha expression in normal colonic mucosa, hyperplastic polyps (HPP), sessile serrated adenomas (SSA), low-grade (TA-LGD) and high-grade (TA-HGD) traditional adenomas as well as in non-metastatic and metastatic colorectal adenocarcinomas. Eight colorectal carcinoma cell lines are tested for their HIF-1alpha inducibility after lipopolysaccharide (LPS) stimulation using western blot and immunocytochemistry. RESULTS: In normal mucosa, HPP and TA-LGD HIF-1alpha was not expressed. In contast, perinuclear protein accumulation and nuclear expression of HIF-1alpha were shown in half of the examined SSA and TA-HGD. In all investigated colorectal carcinomas a significant nuclear HIF-1alpha overexpression compared to the premalignant lesions was observed but a significant correlation with the metastatic status was not found. Nuclear HIF-1alpha expression was strongly accumulated in perinecrotic regions. In these cases HIF-1alpha activation was seen in viable cohesive tumor epithelia surrounding necrosis and in dissociated tumor cells, which subsequently die. Enhanced distribution of HIF-1alpha was also seen in periinflammatory regions. In additional in vitro studies, treatment of diverse colorectal carcinoma cell lines with the potent pro-inflammatory factor lipopolysaccharide (LPS) led to HIF-1alpha expression and nuclear translocation. CONCLUSION: We conclude that HIF-1alpha expression occurs in early stages of colorectal carcinogenesis and achieves a maximum in the invasive stage independent of the metastatic status. Perinecrotic activation of HIF-1alpha in invasive tumors underlines a dual role of HIF-1alpha by regulating both pro-survival and pro-death processes. HIF-1alpha up-regulation in response to LPS-mediated stimulation and periinflammatory expression in invasive carcinomas suggest its involvement in inflammatory events. These patterns of HIF-1alpha inducibility could contribute indirectly to the acquisition of a metastatic phenotype.