CD34 marks angiogenic tip cells in human vascular endothelial cell cultures

The functional shift of quiescent endothelial cells into tip cells that migrate and stalk cells that proliferate is a key event during sprouting angiogenesis. We previously showed that the sialomucin CD34 is expressed in a small subset of cultured endothelial cells and that these cells extend filopodia: a hallmark of tip cells in vivo. In the present study, we characterized endothelial cells expressing CD34 in endothelial monolayers in vitro. We found that CD34-positive human umbilical vein endothelial cells show low proliferation activity and increased mRNA expression of all known tip cell markers, as compared to CD34-negative cells. Genome-wide mRNA profiling analysis of CD34-positive endothelial cells demonstrated enrichment for biological functions related to angiogenesis and migration, whereas CD34-negative cells were enriched for functions related to proliferation. In addition, we found an increase or decrease of CD34-positive cells in vitro upon exposure to stimuli that enhance or limit the number of tip cells in vivo, respectively. Our findings suggest cells with virtually all known properties of tip cells are present in vascular endothelial cell cultures and that they can be isolated based on expression of CD34. This novel strategy may open alternative avenues for future studies of molecular processes and functions in tip cells in angiogenesis

The clinical potential of antiangiogenic fragments of extracellular matrix proteins

Neovasculature development is a crucial step in the natural history of a cancer. While much emphasis has been placed on proangiogenic growth factors such as VEGF, it is clear that endogenous angiogenesis inhibitors also have critical roles in the regulation of this process. Recent research has identified several cryptic fragments of extracellular matrix/vascular basement membrane proteins that have potent antiangiogenic properties in vivo. It has become apparent that many of these fragments signal via interactions with endothelial integrins, although multiple downstream effector pathways have been implicated and endostatin, the first non-collagenous domain of collagen XVIII, influences an intricate signalling network. The activity of these molecules in animal models suggests that they may have significant clinical activity; however, results of phase I/II trials with endostatin were disappointing. Many possible reasons can be found for the failure of these studies. Weaknesses in trial design, endostatin administration regimen and patient selection are identifiable, and importantly the lack of a clearly defined antiangiogenic mechanism for endostatin hindered assessment of biologically effective dose. Additionally, in vivo immunological and proteolytic function-neutralising mechanisms may have negated endostatin's actions. Lessons learned from these studies will aid the future clinical development of other antiangiogenic extracellular matrix protein fragments

Cell death induced by mechanical compression on engineered muscle results from a gradual physiological mechanism

\u3cp\u3eDeep tissue injury (DTI), a type of pressure ulcer, arises in the muscle layers adjacent to bony prominences due to sustained mechanical loading. DTI presents a serious problem in the clinic, as it is often not visible until reaching an advanced stage. One of the causes can be direct mechanical deformation of the muscle tissue and cell. The mechanism of cell death induced by mechanical compression was studied using bio-artificial skeletal muscle tissues. Compression was applied by placing weights on top of the constructs. The morphological changes of the cytoskeleton and the phosphorylation of mitogen-activated protein kinases (MAPK) under compression were investigated. Moreover, inhibitors for each of the three major MAPK groups, p38, ERK, and JNK, were applied separately to look at their roles in the compression caused apoptosis and necrosis. The present study for the first time showed that direct mechanical compression activates MAPK phosphorylation. Compression also leads to a gradual destruction of the cytoskeleton. The percentage apoptosis is strongly reduced by p38 and JNK inhibitors down to the level of the unloaded group. This phenomenon could be observed up to 24. h after initiation of compression. Therefore, cell death in bio-artificial muscle tissue caused by mechanical compression is primarily caused by a physiological mechanism, rather than through a physical mechanism which kills the cell directly. These findings reveal insight of muscle cell death under mechanical compression. Moreover, the result indicates a potential clinical solution to prevent DTI by pre-treating with p38 or/and JNK inhibitors.\u3c/p\u3

Dissecting healthy and diseased hearts

Poster no abstract available

Effect of culture conditions on endothelial cell growth and responsiveness

The in vitro culture of endothelial cells (EC) is dependent on the presence of a coated surface and the availability of growth factors in the medium. The aim of the present research is to investigate whether in vitro EC culture conditions, such as serum source and surface coating, determine the growth characteristics of EC. The phenotype of EC was studied at the level of adhesion molecule expression and down-regulation by angiogenic factors. We found that human umbilical vein EC adhere well to and stretch well with plastic coated with fibronectin, collagen, gelatin and hyaluronan in contrast to non-coated plastic. While low in hyaluronan-coated wells, the spontaneous proliferation of EC was enhanced in fibronectin-collagen and gelatin-coated wells as compared to non-coated wells. Basic fibroblast growth factor bFGF-induced proliferation, however, was best on hyaluronan-coated plastic. A markedly up-regulated proliferation was measured on fibronectin and collagen while EC on gelatin-coated plastic only showed moderate bFGF-induced proliferation. On non-coated plastic EC were not inducible with bFGF. The induction of apoptosis by serum deprivation on these different matrices was most efficient when no coat was available or when wells were coated with hyaluronan, and bFGF inhibited apoptosis induction under all conditions. The use of different culture media demonstrated that human and bovine serum both can be used for human EC assays. The synthetic medium Utroser G prevented both spontaneous and growth factor-induced proliferation. We found that apart from some magnitude differences, the down-regulation of intercellular adhesion molecule-1 (ICAM-1) by angiogenic factors such as bFGF is not dependent on specific culture conditions

Absence of lymphangiogenesis in ductal breast cancer at the primary tumor site

Solid evidence for a relationship between lymphangiogenesis and prognosis in human breast cancer is still lacking. Evidence for ongoing lymphangiogenesis in breast cancer is only provided by animal studies. In the present study we investigated lymphatic vessel density as well as the expression level of the lymphangiogenic factors VEGF-C and -D in a series of 121 ductal breast cancer tissues using immunohistochemical stainings. We found that in the primary tumors the lymphatic vessel density, as well as the expression of both VEGF-C and -D, did not relate to grade, tumor stage, progression or patient survival. Furthermore, in tumors in which lymphatic vessels were present, a Ki-67/podoplanin double staining indicated the absence of proliferating lymphatic endothelial cells. In contrast, we did find a correlation between intratumoral lymphatic vessel density inside the lymph node metastases and patient survival. Another parameter that revealed prognostic value was the presence of tumor cells within the lymphatic vessels. This parameter did predict survival in patients with an age below 63 only. Interestingly, expression of VEGF-D was found to be related to the presence of intralymphatic tumor cells

Essential environmental cues from the satellite cell niche: Optimizing proliferation and differentiation

The use of muscle progenitor cells (MPCs) for regenerative medicine has been severely compromised by their decreased proliferative and differentiative capacity after being cultured in vitro. We hypothesized the loss of pivotal niche factors to be the cause. Therefore, we investigated the proliferative and differentiative response of passage 0 murine MPCs to varying substrate elasticities and protein coatings and found that proliferation was influenced only by elasticity, whereas differentiation was influenced by both elasticity and protein coating. A stiffness of 21 kPa optimally increased the proliferation of MPCs. Regarding differentiation, we demonstrated that fusion of MPCs into myotubes takes place regardless of elasticity. However, ongoing maturation with cross-striations and contractions occurred only on elasticities higher than 3 kPa. Furthermore, maturation was fastest on poly-D-lysine and laminin coatings. Copyrigh