Behavior of CMPCs in unidirectional constrained and stress-free 3D hydrogels.

Cardiomyocyte progenitor cells (CMPCs) are a candidate cell source for cardiac regenerative therapy. However, like other stem cells, after transplantation in the heart, cell retention and differentiation capacity of the CMPCs are low. Combining cells with biomaterials might overcome this problem. By serving as a (temporal) environment, the biomaterial can retain the cells and provide signals that enhance survival, proliferation and differentiation of the cells. To gain more insight into the effect that the encapsulation of CMPCs in a biomaterial has on their behavior, we cultured CMPCs in unidirectional constrained and stress-free collagen/Matrigel hydrogels. CMPCs cultured in 3D hydrogels stay viable and keep their cardiomyogenic profile independent of the application of strain. Moreover, the increased expression of Nkx2.5, myocardin and cTnT in 3D hydrogels compared to 2D cultures, suggests enhanced cardiomyogenic differentiation capacity of cells in 3D. Furthermore, increased expression of collagen I, collagen III, elastin and fibronectin and of the matrix remodeling enzymes MMP-1, MMP-2, MMP-9, and TIMP-1 and TIMP-2 in the 3D hydrogels is indicative of an enhanced matrix remodeling capacity of CMPCs in a 3D environment, independent of the application of strain. Interestingly, the additional application of static strain to the 3D hydrogels, as imposed by hydrogel constrainment, stabilized CMPC viability and proliferation, resulted in enhanced cardiac marker protein expression and appeared crucial for cellular organization and morphology. More specifically, CMPCs cultured in 3D collagen/Matrigel constrained hydrogels became readily mechanosensitive, had a rod-shaped morphology, and responded to the applied strain by orienting in the direction of the constraint. Overall, our data demonstrate the applicability of CMPCs in a 3D environment since encapsulation of CMPCs may stabilize survival and proliferation, can enhance the differentiation and remodeling capacity of the cells, and could induce cellular re-organization, which all may contribute to an improved efficiency of cardiac stem cell therapy.

Matrix production and remodeling capacity of cardiomyocyte progenitor cells during in vitro differentiation.

Cell-based therapy has emerged as a treatment modality for myocardial repair. Especially cardiac resident stem cells are considered a potential cell source since they are able to differentiate into cardiomyocytes and have improved heart function after injury in a preclinical model for myocardial infarction. To avoid or repair myocardial damage it is important not only to replace the lost cardiomyocytes, but also to remodel and replace the scar tissue by "healthy" extracellular matrix (ECM). Interestingly, the role of cardiac stem cells in this facet of cardiac repair is largely unknown. Therefore, we investigated the expression and production of ECM proteins, matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) in human cardiomyocyte progenitor cells (CMPCs) undergoing differentiation towards the cardiomyogenic lineage. Our data suggest that CMPCs have the capacity to synthesize and modulate their own matrix environment, especially during differentiation towards the cardiomyogenic lineage. While undifferentiated CMPCs expressed collagen I, III, IV and fibronectin, but no elastin, during the process of differentiation the expression of collagen I, III, IV and fibronectin increased and interestingly also elastin expression was induced. Furthermore, undifferentiated CMPCs express MMP-1 -2 and -9 and upon differentiation the expression of MMP-1 decreased, while the expression of MMP-2 and MMP-9, although the latter only in the early stage of differentiation, increased. Additionally, the expression of TIMP-1, -2 and -4 was induced during differentiation. This study provides new insights into the matrix production and remodeling capacity of human CMPCs, with potential beneficial effects for the treatment of cardiac injury.

Multi-parametric assessment of the anti-angiogenic effects of liposomal glucocorticoids.

Inflammation plays a prominent role in tumor growth. Anti-inflammatory drugs have therefore been proposed as anti-cancer therapeutics. In this study, we determined the anti-angiogenic activity of a single dose of liposomal prednisolone phosphate (PLP-L), by monitoring tumor vascular function and viability over a period of one week. C57BL/6 mice were inoculated subcutaneously with B16F10 melanoma cells. Six animals were PLP-L-treated and six served as control. Tumor tissue and vascular function were probed using MRI before and at three timepoints after treatment. DCE-MRI was used to determine K(trans), v(e), time-to-peak, initial slope and the fraction of non-enhancing pixels, complemented with immunohistochemistry. The apparent diffusion coefficient (ADC), T(2) and tumor size were assessed with MRI as well. PLP-L treatment resulted in smaller tumors and caused a significant drop in K(trans) 48 h post-treatment, which was maintained until one week after drug administration. However, this effect was not sufficient to significantly distinguish treated from non-treated animals. The therapy did not affect tumor tissue viability but did prevent the ADC decrease observed in the control group. No evidence for PLP-L-induced tumor vessel normalization was found on histology. Treatment with PLP-L altered tumor vascular function. This effect did not fully explain the tumor growth inhibition, suggesting a broader spectrum of PLP-L activities.

Synergistic targeting of alphavbeta3 integrin and galectin-1 with heteromultivalent paramagnetic liposomes for combined MR imaging and treatment of angiogenesis.

Effective and specific targeting of nanoparticles is of paramount importance in the fields of targeted therapeutics and diagnostics. In the current study, we investigated the targeting efficacy of nanoparticles that were functionalized with two angiogenesis-specific targeting ligands, an alpha(v)beta(3) integrin-specific and a galectin-1-specific peptide. We show in vitro, using optical techniques and MRI, that the dual-targeting approach produces synergistic targeting effects, causing a dramatically elevated uptake of nanoparticles as compared to single ligand targeting.

Paramagnetic and fluorescent liposomes for target-specific imaging and therapy of tumor angiogenesis.

Angiogenesis is essential for tumor growth and metastatic potential and for that reason considered an important target for tumor treatment. Noninvasive imaging technologies, capable of visualizing tumor angiogenesis and evaluating the efficacy of angiostatic therapies, are therefore becoming increasingly important. Among the various imaging modalities, magnetic resonance imaging (MRI) is characterized by a superb spatial resolution and anatomical soft-tissue contrast. Revolutionary advances in contrast agent chemistry have delivered versatile angiogenesis-specific molecular MRI contrast agents. In this paper, we review recent advances in the preclinical application of paramagnetic and fluorescent liposomes for noninvasive visualization of the molecular processes involved in tumor angiogenesis. This liposomal contrast agent platform can be prepared with a high payload of contrast generating material, thereby facilitating its detection, and is equipped with one or more types of targeting ligands for binding to specific molecules expressed at the angiogenic site. Multimodal liposomes endowed with contrast material for complementary imaging technologies, e.g., MRI and optical, can be exploited to gain important preclinical insights into the mechanisms of binding and accumulation at angiogenic vascular endothelium and to corroborate the in vivo findings. Interestingly, liposomes can be designed to contain angiostatic therapeutics, allowing for image-supervised drug delivery and subsequent monitoring of therapeutic efficacy.

Cellular compartmentalization of internalized paramagnetic liposomes strongly influences both T1 and T2 relaxivity.

In recent years, numerous Gd(3+)-based contrast agents have been developed to enable target-specific MR imaging of in vivo processes at the molecular level. The combination of powerful contrast agents and amplification strategies, aimed at increasing the contrast agent dose at the target site, is an often-used strategy to improve the sensitivity of biomarker detection. One such amplification mechanism is to target a disease-specific cell membrane receptor that can undergo multiple rounds of internalization following ligand binding and thus shuttle a sizeable amount of contrast agent into the target cell. An example of such a membrane receptor is the alpha(nu)beta(3) integrin. The goal of this study was to investigate the consequences of this amplification approach for the T(1)- and T(2)-shortening efficacy of a paramagnetic contrast agent. Cultured endothelial cells were incubated with paramagnetic liposomes that were conjugated with a cyclic RGD-peptide to enable internalization by means of the alpha(nu)beta(3) integrin receptor. Non-targeted liposomes served as a control. This study showed that alpha(nu)beta(3) targeting dramatically increased the uptake of paramagnetic liposomes. This targeting strategy, however, strongly influenced both the longitudinal and transverse relaxivity of the internalized paramagnetic liposomes.

Early in vivo assessment of angiostatic therapy efficacy by molecular MRI.

Noninvasive diagnostic imaging methods to establish the efficacy of angiostatic therapies are becoming increasingly important with the first Food and Drug Administration approvals of such agents. Magnetic resonance molecular imaging is an imaging technique that allows the visualization of pathological processes in vivo with a better spatial resolution as compared with nuclear methods, such as photon emission tomography and single photon emission computed tomography. In this study, we used alpha(v)beta3 targeted bimodal liposomes to quantitate angiogenesis in a tumor mouse model with magnetic resonance imaging (MRI) and to evaluate the therapeutic efficacy of the angiogenesis inhibitors anginex and endostatin. The MRI findings were validated with fluorescence microscopy and showed a very good correlation with the microvessel density. In conclusion, this study provides evidence that molecular MRI can be used to noninvasively measure the efficacy of angiogenesis inhibitors during the course of therapy.

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.

Anti-angiogenesis therapy can overcome endothelial cell anergy and promote leukocyte-endothelium interactions and infiltration in tumors.

Tumor escape from immunity, as well as the failure of several anti-cancer vaccination and cellular immunotherapy approaches, is suggested to be due to the angiogenesis-mediated suppression of endothelial cell (EC) adhesion molecules involved in leukocyte-vessel wall interactions. We hypothesized that inhibition of angiogenesis would overcome this escape from immunity. We investigated this in vivo by means of intravital microscopy and ex vivo by immunohistochemistry in two mouse tumor models. Angiogenesis inhibitors anginex, endostatin, and angiostatin, and the chemotherapeutic agent paclitaxel were found to significantly stimulate leukocyte-vessel wall interactions by circumvention of EC anergy in vivo, i.e., by the up-regulation of endothelial adhesion molecules in tumor vessels. This was confirmed by in vitro studies of cultured EC at the protein and mRNA levels. The new angiostatic designer peptide anginex was most potent at overcoming EC anergy; the enhanced leukocyte-vessel interactions led to an increase in the numbers of tumor infiltrating leukocytes. While anginex inhibited tumor growth and microvessel density significantly, the amount of infiltrated leukocytes (CD45), as well as the number of CD8+ cytotoxic T lymphocytes, was enhanced markedly. The current results suggest that immunotherapy strategies can be improved by combination with anti-angiogenesis.

Tumor cell plasticity in Ewing sarcoma, an alternative circulatory system stimulated by hypoxia.

A striking feature of Ewing sarcoma is the presence of blood lakes lined by tumor cells. The significance of these structures, if any, is unknown. Here, we report that the extent of blood lakes correlates with poor clinical outcomes, whereas variables of angiogenesis do not. We also show that Ewing sarcoma cells form vessel-like tubes in vitro and express genes associated with vasculogenic mimicry. In tumor models, we show that there is blood flow through the blood lakes, suggesting that these structures in Ewing sarcoma contribute to the circulation. Furthermore, we present evidence that reduced oxygen tension may be instrumental in tube formation by plastic tumor cells. The abundant presence of these vasculogenic structures, in contrast to other tumor types, makes Ewing sarcoma the ideal model system to study these phenomena. The results suggest that optimal tumor treatment may require targeting of these structures in combination with prevention of angiogenesis.

MR molecular imaging and fluorescence microscopy for identification of activated tumor endothelium using a bimodal lipidic nanoparticle.

In oncological research, there is a great need for imaging techniques that specifically identify angiogenic blood vessels in tumors on the basis of differences in the expression level of biomolecular markers. In the angiogenic cascade, different cell surface receptors, including the alphavbeta3-integrin, are strongly expressed on activated endothelial cells. In the present study, we aimed to image angiogenesis by detecting the expression of alphavbeta3 in tumor bearing mice with a combination of magnetic resonance imaging (MRI) and fluorescence microscopy. To that end, we prepared MR-detectable and fluorescent liposomes, which carry approximately 700 alphavbeta3-specific RGD peptides per liposome. RGD competition experiments and RAD-conjugated liposomes were used as controls for specificity. In vivo, both RAD liposomes and RGD liposomes gave rise to signal increase on T1-weighted MR images. It was established by the use of ex vivo fluorescence microscopy that RGD liposomes and RAD liposomes accumulated in the tumor by different mechanisms. RGD liposomes were specifically associated with activated tumor endothelium, while RAD liposomes were located in the extravascular compartment. This study demonstrates that MR molecular imaging of angiogenesis is feasible by using a targeted contrast agent specific for the alphavbeta3-integrin, and that the multimodality imaging approach gave insight into the exact mechanism of accumulation in the tumor.

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

Deep 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 24h 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.

The designer anti-angiogenic peptide anginex targets tumor endothelial cells and inhibits tumor growth in animal models.

The de novo designed angiogenesis inhibitor anginex was tested in vitro and in vivo for its mechanism of action and antitumor activity. The data presented here demonstrate that anginex is a powerful antiangiogenic agent with significant antitumor activity. The mechanism of action of anginex was found to be the induction of anoikis leading to apoptosis in angiogenically activated endothelial cells, resulting in an up to 90% inhibition of migration in the wound assay. Anginex inhibited angiogenesis as demonstrated in the in vitro mouse aortic ring assay. In addition, tumor-induced angiogenesis in the chick chorioallantoic membrane was markedly inhibited. Anginex showed profound antitumor activity in the syngeneic mouse B16F10 melanoma model and in a xenograft human tumor model. Microvessel density determination as well as magnetic resonance imaging showed that the antitumor activity in these tumor models resulted from the antiangiogenic activity of anginex. A complete absence of toxicity was observed in these models. The data presented here demonstrate that anginex is a promising agent for further clinical development.

CD44 enhances tumor aggressiveness by promoting tumor cell plasticity.

Aggressive tumor cells can obtain the ability to transdifferentiate into cells with endothelial features and thus form vasculogenic networks. This phenomenon, called vasculogenic mimicry (VM), is associated with increased tumor malignancy and poor clinical outcome. To identify novel key molecules implicated in the process of vasculogenic mimicry, microarray analysis was performed to compare gene expression profiles of aggressive (VM+) and non-aggressive (VM-) cells derived from Ewing sarcoma and breast carcinoma. We identified the CD44/c-Met signaling cascade as heavily relevant for vasculogenic mimicry. CD44 was at the center of this cascade, and highly overexpressed in aggressive tumors. Both CD44 standard isoform and its splice variant CD44v6 were linked to increased aggressiveness in VM. Since VM is most abundant in Ewing sarcoma tumors functional analyses were performed in EW7 cells. Overexpression of CD44 allowed enhanced adhesion to its extracellular matrix ligand hyaluronic acid. CD44 expression also facilitated the formation of vasculogenic structures in vitro, as CD44 knockdown experiments repressed migration and vascular network formation. From these results and the observation that CD44 expression is associated with vasculogenic structures and blood lakes in human Ewing sarcoma tissues, we conclude that CD44 increases aggressiveness in tumors through the process of vasculogenic mimicry.

Anti-tumor activity of the novel angiogenesis inhibitor anginex.

Anginex is a novel cytokine-like peptide with potent anti-angiogenic activity, which operates specifically against angiogenically-activated endothelial cells via prevention of cell adhesion/migration on the extracellular matrix and subsequent induction of apoptosis. Here, we demonstrate that anginex inhibits tumor growth in vivo in mouse xenograft models. In the MA148 ovarian carcinoma model, tumor growth was inhibited dose-dependently by up to 80% when systemically administered via osmotic mini-pumps starting at the time of tumor cell inoculation. The optimal dose was found to be 10 mg/kg per day. When tested against established tumors, mini-pump-administered anginex demonstrated essentially the same effectivity at this optimal dose, whereas once or twice-daily injections were only half as effective. When anginex was conjugated to human serum albumin, effectivity was significantly improved, most likely due to increased bioavailability of the conjugate. Immunohistochemical analysis of microvessel density indicated that the anti-tumor activity of anginex is mediated by angiogenesis inhibition. This was confirmed in an in vitro angiogenesis assay based on tube formation in a collagen gel. Animals demonstrated no signs of toxicity as judged by unaltered behavior, normal weight gain, blood markers and macro- and microscopic morphology of internal organs upon autopsy. Overall, these in vivo studies indicate that anginex is an effective anti-tumor agent.

Material-based engineering strategies for cardiac regeneration.

Cardiac tissue is composed of muscle and non-muscle cells, surrounded by extracellular matrix (ECM) and spatially organized into a complex three-dimensional (3D) architecture to allow for coordinated contraction and electrical pulse propagation. Despite emerging evidence for cardiomyocyte turnover in mammalian hearts, the regenerative capacity of human cardiac tissue is insufficient to recover from damage, e.g. resulting from myocardial infarction (MI). Instead, the heart 'repairs' lost or injured tissue by ongoing synthesis and remodeling of scar tissue. Conventional therapies and timely (stem) cell delivery to the injured tissue markedly improve short-term function and remodeling, but do not attenuate later stage adverse remodeling, leading to functional deterioration and eventually failure of the heart. Material-based therapies have been successfully used to mechanically support and constrain the post-MI failing heart, preventing it from further remodeling and dilation. When designed to deliver the right microenvironment for endogenous or exogenous cells, as well as the mechanical and topological cues to guide neo-tissue formation, material-based therapies may even reverse remodeling and boost cardiac regeneration. This paper reviews the up-to-date status of material-based cardiac regeneration with special emphasis on 1) the use of bare biomaterials to deliver passive constraints that unload the heart, 2) the use of materials and cells to create engineered cardiac constructs for replacement, support, or regeneration of damaged myocardium, and 3) the development of bio-inspired and bioactive materials that aim to enhance the endogenous regenerative capacity of the heart. As the therapies should function in the infarcted heart, the damaged host environment and engineered in vitro test systems that mimic this environment, are reviewed as well.

Supramolecular control of cell adhesion via ferrocene-cucurbit[7]uril host-guest binding on gold surfaces.

Supramolecular control of adhesion of cells is demonstrated using synthetic integrin binding RGD peptide-ferrocene conjugates that were immobilized via host-guest chemistry onto cucurbit[7]uril coated gold surfaces.

Engineering skeletal muscle tissues from murine myoblast progenitor cells and application of electrical stimulation.

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to study pressure ulcers, for regenerative medicine and as a meat alternative (1). The first reported 3D muscle constructs have been made many years ago and pioneers in the field are Vandenburgh and colleagues (2,3). Advances made in muscle tissue engineering are not only the result from the vast gain in knowledge of biochemical factors, stem cells and progenitor cells, but are in particular based on insights gained by researchers that physical factors play essential roles in the control of cell behavior and tissue development. State-of-the-art engineered muscle constructs currently consist of cell-populated hydrogel constructs. In our lab these generally consist of murine myoblast progenitor cells, isolated from murine hind limb muscles or a murine myoblast cell line C2C12, mixed with a mixture of collagen/Matrigel and plated between two anchoring points, mimicking the muscle ligaments. Other cells may be considered as well, e.g. alternative cell lines such as L6 rat myoblasts (4), neonatal muscle derived progenitor cells (5), cells derived from adult muscle tissues from other species such as human (6) or even induced pluripotent stem cells (iPS cells) (7). Cell contractility causes alignment of the cells along the long axis of the construct (8,9) and differentiation of the muscle progenitor cells after approximately one week of culture. Moreover, the application of electrical stimulation can enhance the process of differentiation to some extent (8). Because of its limited size (8 x 2 x 0.5 mm) the complete tissue can be analyzed using confocal microscopy to monitor e.g. viability, differentiation and cell alignment. Depending on the specific application the requirements for the engineered muscle tissue will vary; e.g. use for regenerative medicine requires the up scaling of tissue size and vascularization, while to serve as a meat alternative translation to other species is necessary.

MicroRNA-1 and microRNA-206 improve differentiation potential of human satellite cells: a novel approach for tissue engineering of skeletal muscle.

Innovative strategies based on regenerative medicine, in particular tissue engineering of skeletal muscle, are promising for treatment of patients with skeletal muscle damage. However, the efficiency of satellite cell differentiation in vitro is suboptimal. MicroRNAs are involved in the regulation of cell proliferation and differentiation. We hypothesized that transient overexpression of microRNA-1 or microRNA-206 enhances the differentiation potential of human satellite cells by downregulation quiescent satellite cell regulators, thereby increasing myogenic regulator factors. To investigate this, we isolated and cultured human satellite cells from muscle biopsies. First, through immunofluorescent analysis and quantitative reverse transcription-polymerase chain reaction (qRT-PCR), we showed that in satellite cell cultures, low Pax7 expression is related to high MyoD expression on differentiation, and, subsequently, more extensive sarcomere formation, that is, muscle differentiation, was detected. Second, using qRT-PCR, we showed that microRNA-1 and microRNA-206 are robustly induced in differentiating satellite cells. Finally, a gain-of-function approach was used to investigate microRNA-1 and microRNA-206 potential in human satellite cells to improve differentiation potential. As a proof of concept, this was also investigated in a three-dimensional bioartificial muscle construct. After transfection with microRNA-1, the number of Pax7 expressing cells decreased compared with the microRNA-scrambled control. In differentiated satellite cell cultures transfected with either microRNA-1 or microRNA-206, the number of MyoD expressing cells increased, and α-sarcomeric actin and myosin expression increased compared with microRNA-scrambled control cultures. In addition, in a three-dimensional bioartificial muscle construct, an increase in MyoD expression occurred. Therefore, we conclude that microRNA-1 and microRNA-206 can improve human satellite cell differentiation. It represents a potential novel approach for tissue engineering of human skeletal muscle for the benefit of patients with facial paralysis.

Mechanoregulation of vascularization in aligned tissue-engineered muscle: a role for vascular endothelial growth factor.

Skeletal muscle tissue engineering has major promise for regenerative treatment of patients suffering from muscle loss due to, for example, traumatic injury, but faces considerable challenges to progress toward clinical application. In the present study the creation of an aligned prevascularized muscle tissue was addressed. We hypothesized that an aligned vascularized three-dimensional (3D) muscle tissue can be induced in vitro by merely using uniaxial stress. The present study showed that not only do endothelial cells and muscle cells independently align in the direction of uniaxial stress in a hydrogel-based 3D culture system, but also, more importantly, the endothelial cells in the co-cultured 3D constructs organized into vascular structures. Strikingly, in these cultures no additional growth factors were needed to induce vascular formation of the endothelial cells. Vascular endothelial growth factor (VEGF) production by the muscle cells was stimulated by the uniaxial stress that develops in the tissue when constrained in one direction. This stress accompanied by VEGF production appeared to play a key role in the organization of the endothelial cells into vessel-like structures.