A mathematical model of integrin-mediated haptotactic cell migration

Haptotactic cell migration, a directed response to gradients of cell—extracellular matrix adhesion, is an important process in a number of biological phenomena such as wound healing and tumour cell invasion. Previously, mathematical models of haptotaxis have been developed on the premise that cells migrate in response to gradients in the density of the extracellular matrix. In this paper, we develop a novel mathematical model of haptotaxis which includes the adhesion receptors known as integrins and a description of their functional activation, local recruitment and protrusion as part of lamellipodia. Through the inclusion of integrins, the modelled cell matter is able to respond to a true gradient of cell–matrix adhesion, represented by functionally active integrins. We also show that previous matrix-mediated models are in fact a subset of the novel integrin-mediated models, characterised by specific choices of diffusion and haptotaxis coefficients in their model equations. Numerical solutions suggest the existence of travelling waves of cell migration that are confirmed via a phase plane analysis of a simplified model.

Modelling oxygen diffusion and cell growth in a porous, vascularising scaffold for soft tissue engineering applications

Soft tissue engineering presents significant challenges compared to other tissue engineering disciplines such as bone, cartilage or skin engineering. The very high cell density in most soft tissues, often combined with large implant dimensions, means that the supply of oxygen is a critical factor in the success or failure of a soft tissue scaffold. A model is presented for oxygen diffusion in a 15-60 mm diameter dome-shaped scaffold fed by a blood vessel loop at its base. This model incorporates simple models for vascular growth, cell migration and the effect of cell density on the effective oxygen diffusivity. The model shows that the dynamic, homogeneous cell seeding method often employed in small-scale applications is not applicable in the case of larger scale scaffolds such as these. Instead, we propose the implantation of a small biopsy of tissue close to a blood supply within the scaffold as a technique more likely to be successful. Crown Copyright (c) 2005 Published by Elsevier Ltd. All rights reserved.

Robo1 regulates the development of major axon tracts and interneuron migration in the forebrain

The Slit genes encode secreted ligands that regulate axon branching, commissural axon pathfinding and neuronal migration. The principal identified receptor for Slit is Robo ( Roundabout in Drosophila). To investigate Slit signalling in forebrain development, we generated Robo1 knockout mice by targeted deletion of exon 5 of the Robo1 gene. Homozygote knockout mice died at birth, but prenatally displayed major defects in axon pathfinding and cortical interneuron migration. Axon pathfinding defects included dysgenesis of the corpus callosum and hippocampal commissure, and abnormalities in corticothalamic and thalamocortical targeting. Slit2 and Slit1/2 double mutants display malformations in callosal development, and in corticothalamic and thalamocortical targeting, as well as optic tract defects. In these animals, corticothalamic axons form large fasciculated bundles that aberrantly cross the midline at the level of the hippocampal and anterior commissures, and more caudally at the medial preoptic area. Such phenotypes of corticothalamic targeting were not observed in Robo1 knockout mice but, instead, both corticothalamic and thalamocortical axons aberrantly arrived at their respective targets at least 1 day earlier than controls. By contrast, in Slit mutants, fewer thalamic axons actually arrive in the cortex during development. Finally, significantly more interneurons ( up to twice as many at E12.5 and E15.5) migrated into the cortex of Robo1 knockout mice, particularly in both rostral and parietal regions, but not caudal cortex. These results indicate that Robo1 mutants have distinct phenotypes, some of which are different from those described in Slit mutants, suggesting that additional ligands, receptors or receptor partners are likely to be involved in Slit/Robo signalling.

Repelling class discrimination: ephrin-A5 binds to and activates EphB2 receptor signaling

The interactions between Eph receptor tyrosine kinases and their ephrin ligands regulate cell migration and axon pathfinding. The EphA receptors are generally thought to become activated by ephrin-A ligands, whereas the EphB receptors interact with ephrin-B ligands. Here we show that two of the most widely studied of these molecules, EphB2 and ephrin-A5, which have never been described to interact with each other, do in fact bind one another with high affinity. Exposure of EphB2-expressing cells to ephrin-A5 leads to receptor clustering, autophosphorylation and initiation of downstream signaling. Ephrin-A5 induces EphB2-mediated growth cone collapse and neurite retraction in a model system. We further show, using X-ray crystallography, that the ephrin-A5-EphB2 complex is a heterodimer and is architecturally distinct from the tetrameric EphB2-ephrin-B2 structure. The structural data reveal the molecular basis for EphB2-ephrin-A5 signaling and provide a framework for understanding the complexities of functional interactions and crosstalk between A- and B-subclass Eph receptors and ephrins.

Equine laminitis: increased transcription of matrix metalloproteinase-2 (MMP-2) occurs during the developmental phase

Reasons for performing study: The dysadhesion and destruction of lamellar basement membrane of laminitis may be due to increased lamellar metalloproteinase activity. Characterising lamellar metalloproteinase-2 (MMP-2) and locating it in lamellar tissues may help determine if laminitis pathology is associated with increased MMP-2 transcription. Objectives: To clone and sequence the cDNA encoding lamellar MMP-2, develop antibody and in situ hybridisation probes to locate lamellar MMP-2 and quantitate MMP-2 transcription in normal and laminitis tissue. Methods: Total RNA was isolated, fragmented by RT-PCR, cloned into vector and sequenced. Rabbit anti-equine MMP-2 and labelled MMP-2 riboprobe were developed to analyse and quantitate MMP-2 expression. Results: Western immunoblotting with anti-MMP-2 detected 72 kDa MMP-2 in hoof tissue homogenates and cross-reacted with human MMP-2. Immunohistochemistry and in situ hybridisation detected MMP-2 in the cytoplasm of basal and parabasal cells in close proximity to the lamellar basement membrane. Northern analysis and quantitative real-time PCR showed MMP-2 expression significantly (P

Equine laminitis: cleavage of laminin 5 associated with basement membrane dysadhesion

Reasons for performing study: The key lesion of laminitis is separation at the hoof lamellar dermal-epidermal interface. For this to happen the structural and adhesion proteins of the basement membrane zone must be altered. Which proteins and how damage to them leads to the lamellar separation of laminitis is unknown. Objectives: To investigate lamellar hemidesmosome and cytoskeleton damage and basement membrane dysadhesion using light microscopy (LM) and immunofluorescence microscopy (IFM). Methods: Cryostat sections of lamellar tissues from 2 control and 6 Standardbred horses with oligofructose induced laminitis were studied using LM and IFM. Plectin, integrin alpha(6) and BP230 antibody was used to label hemidesmosome intracellular plaque proteins and anti-BP180 and anti-laminin 5 (L5) was used to label anchoring filament (AF) proteins. Cytoskeleton intermediate filaments were labelled using anti-cytokeratin 14. The primary antibodies of selected sections were double labelled to show protein co-localisation. Results: Laminitis caused reduction of transmembrane integrin alpha(6), the AF proteins BP180 and L5,and failure of co-localisation of BP180 and L5. Proteins of the inner hemidesmosomal plaque, plectin and BP230, were unaffected. Conclusions: Loss of co-localisation of L5 and BP180 suggests that, during the acute phase of laminitis, L5 is cleaved and therefore, the AFs connecting the epidermis to the dermis, fail. Without a full complement of AFs separation at the lamellar dermo-epidermal junction occurs. Potential relevance: Suppressing or inhibiting metalloproteinase activity may prevent L5 cleavage and therefore the lamellar dermo-epidermal separation of laminitis.

Kallikrein 4 (hK4) and prostate-specific antigen (PSA) are associated with the loss of E-cadherin and an epithelial-mesenchymal transition (EMT)-like effect in prostate cancer cells

Prostate-specific antigen (PSA) and the related kallikrein family of serine proteases are current or emerging biomarkers for prostate cancer detection and progression. Kallikrein 4 (KLK4/hK4) is of particular interest, as KLK4 mRNA has been shown to be elevated in prostate cancer. In this study, we now show that the comparative expression of hK4 protein in prostate cancer tissues, compared with benign glands, is greater than that of PSA and kallikrein 2 (KLK2/hK2), suggesting that hK4 may play an important functional role in prostate cancer progression in addition to its biomarker potential. To examine the roles that hK4, as well as PSA and hK2, play in processes associated with progression, these kallikreins were separately transfected into the PC-3 prostate cancer cell line, and the consequence of their stable transfection was investigated. PC-3 cells expressing hK4 had a decreased growth rate, but no changes in cell proliferation were observed in the cells expressing PSA or hK2. hK4 and PSA, but not hK2, induced a 2.4-fold and 1.7-fold respective increase, in cellular migration, but not invasion, through Matrigel, a synthetic extracellular matrix. We hypothesised that this increase in motility displayed by the hK4 and PSA-expressing PC-3 cells may be related to the observed change in structure in these cells from a typical rounded epithelial-like cell to a spindle-shaped, more mesenchymal-like cell, with compromised adhesion to the culture surface. Thus, the expression of E-cadherin and vimentin, both associated with an epithelial-mesenchymal transition (EMT), was investigated. E-cadherin protein was lost and mRNA levels were significantly decreased in PC-3 cells expressing hK4 and PSA (10-fold and 7-fold respectively), suggesting transcriptional repression of E-cadherin, while the expression of vimentin was increased in these cells. The loss of E-cadherin and associated increase in vimentin are indicative of EMT and provides compelling evidence that hK4, in particular, and PSA have a functional role in the progression of prostate cancer through their promotion of tumour cell migration.

CXCR4/SDF1 interaction inhibits the primordial to primary follicle transition in the neonatal mouse ovary

The molecular mechanisms behind the entry of the primordial follicle into the growing follicle pool remain poorly understood. To investigate this process further, a microarray-based comparison was undertaken between 2-day postpartum mouse ovaries consisting of primordial follicles/naked oocytes only and those with both primordial follicles and newly activated follicles (7-day postpartum). Gene candidates identified included the chemoattractive cytokine stromal derived factor-1 (SDF1) and its receptor CXCR4. SDF1 and CXCR4 have been implicated in a variety of physiological processes including the migration of embryonic germ cells to the gonads. SDF1-alpha expression increased with the developmental stage of the follicle. Embryonic expression was found to be dichotomous post-genii cell migration, with low expression in the female. Immunohistochemical studies nonetheless indicate that the autocrine pattern of expression ligand and receptor begins during embryonic life. Addition of recombinant SDF1-alpha to neonatal mouse ovaries in vitro resulted in significantly higher follicle densities than for control ovaries. TUNEL analysis indicated no detectable difference in populations of apoptotic cells of treated or control ovaries. Treated ovaries also contained a significantly lower percentage of activated follicles as determined by measurement of oocyte diameter and morphological analysis. Treatment of cultured ovaries with an inhibitor of SDF1-alpha, AMD3100, ablated the effect of SDF1-alpha. By retaining follicles in an unactivated state, SDF1/CXCR4 signaling may play an important role in maintaining the size and longevity of the primordial follicle pool. (c) 2006 Elsevier Inc. All rights reserved.

Effect of disrupted SOX18 transcription factor function on tumor growth, vascularization, and endothelial development

Background. The growth of solid tumors depends on establishing blood supply; thus, inhibiting tumor angiogenesis has been a long-term goal in cancer therapy. The SOX18 transcription factor is a key regulator of murine and human blood vessel formation. Methods: We established allograft melanoma tumors in wild-type mice, Sox18-null mice, and mice expressing a dominant-negative form of Sox18 (Sox18RaOp) (n = 4 per group) and measured tumor growth and microvessel density by immunohistochemical analysis with antibodies to the endothelial marker CD31 and the pericyte marker NG2. We also assessed the affects of disrupted SOX18 function on MCF-7 human breast cancer and human umbilical vein endothelial cell (HUVEC) proliferation by measuring BrdU incorporation and by MTS assay, cell migration using Boyden chamber assay, and capillary tube formation in vitro. All statistical tests were two-sided. Results: Allograft tumors in Sox18-null and Sox18RaOp mice grew more slowly than those in wild-type mice (tumor volume at day 14, Sox18 null, mean = 486 mm(3), 95% confidence interval [CI] = 345 mm(3) to 627 mm(3), p = .004; Sox18RaOp, mean = 233 mm(3), 95% CI = 73 mm(3) to 119 mm(3), p < .001; versus wild-type, mean = 817 mm(3), 95% CI = 643 mm(3) to 1001 mm(3)) and had fewer CD31- and NG2-expressing vessels. Expression of dominant-negative Sox18 reduced the proliferation of MCF-7 cells (BrdU incorporation: MCF-7(Ra) = 20%, 95% CI = 15% to 25% versus MCF-7 = 41%, 95% CI = 35% to 45%; P = .013) and HUVECs (optical density at 490 nm, empty vector, mean = 0.46 versus SOX18 mean = 0.29; difference = 0.17, 95% CI = 0.14 to 0.19; P = .001) compared with control subjects. Overexpression of wild-type SOX18 promoted capillary tube formation of HUVECs in vitro, whereas expression of dominant-negative SOX18 impaired tube formation of HUVECs and the migration of MCF-7 cells via the disruption of the actin cytoskeleton. Conclusions: SOX18 is a potential target for antiangiogenic therapy of human cancers.

Localization of neogenin protein during morphogenesis in the mouse embryo

Neogenin, a close relative of the axon guidance receptor DCC, has been shown to be a receptor for members of the Netrin and Repulsive Guidance Molecule families. Recent studies have begun to uncover a role for Neogenin in organogenesis. Here we examine the localization of Neogenin protein in the developing mouse embryo (embryonic day 14.5) when organogenesis is progressing rapidly. We observe that Neogenin protein is restricted to distinct tissue layers within a given organ. In some embryonic epithelia such as the gut and pancreas, Neogenin protein is predominantly polarized to the basal surfaces of the epithelial cells. In contrast, Neogenin is restricted to mesenchymal cells within the lung and kidney. Neogenin is also seen in differentiating skeletal muscle and condensing cartilage throughout the embryo, and in the trigeminal and dorsal root ganglia of the peripheral nervous system. This study supports the emerging role for Neogenin as a key receptor in the establishment of the morphological architecture in many developing organ systems.

Overexpression of transforming growth factor-beta is associated with increased hyaluronan content and impairment of repair in Marfan syndrome aortic aneurysm

Background-Marfan syndrome (MFS), a condition caused by fibrillin-1 gene mutation is associated with aortic aneurysm that shows elastic lamellae disruption, accumulation of glycosaminoglycans, and vascular smooth muscle cell (VSMC) apoptosis with minimal inflammatory response. We examined aneurysm tissue and cultured cells for expression of transforming growth factor-beta1 to -beta3 (TGF beta 1 to 3), hyaluronan content, apoptosis, markers of cell migration, and infiltration of vascular progenitor cells (CD34). Methods and Results-MFS aortic aneurysm (6 males, 5 females; age 8 to 78 years) and normal aorta (5 males, 3 females; age 22 to 56 years) were used. Immunohistochemistry showed increased expression of TGF beta 1 to 3, hyaluronan, and CD34-positive microcapillaries in MFS aneurysm compared with control. There was increased expression of TGF beta 1 to 3 and hyaluronan in MFS cultured VSMCs, adventitial fibroblasts (AF), and skin fibroblasts (SF). Apoptosis was increased in MFS (VSMC: mean cell loss in MFS 29%, n of subjects = 5, versus control 8%, n = 3, P < 0.05; AF: 28%, n = 5 versus 7%, n = 5, P < 0.05; SF: 29%, n = 3 versus 4%, n = 3, not significant). In MFS, there was a 2-fold increase in adventitial microcapillaries containing CD34-positive cells compared with control tissue. Scratch wound assay showed absence of CD44, MT1-MMP, and beta-3 integrin at the leading edge of migration in MFS indicating altered directional migration. Western blot showed increased expression of TGF beta 1 to 3 in MFS but no change in expression of CD44, MT1-MMP, or beta-3 integrin compared with controls. Conclusions-There was overexpression of TGF-beta in MFS associated with altered hyaluronan synthesis, increased apoptosis, impaired progenitor cell recruitment, and abnormal directional migration. These factors limit tissue repair and are likely to contribute to aneurysm development.

The makings of maleness: towards an integrated view of male sexual development

As the mammalian embryo develops, it must engage one of the two distinct programmes of gene activity, morphogenesis and organogenesis that characterize males and females. In males, sexual development hinges on testis determination and differentiation, but also involves many coordinated transcriptional, signalling and endocrine networks that underpin the masculinization of other organs and tissues, including the brain. Here we bring together current knowledge about these networks, identify gaps in the overall picture, and highlight the known defects that lead to disorders of male sexual development.

Method for generation of homogeneous multicellular tumor spheroids applicable to a wide variety of cell types

Multicellular tumor spheroids (MCTS) are used as organotypic models of normal and solid tumor tissue. Traditional techniques for generating MCTS, such as growth on nonadherent surfaces, in suspension, or on scaffolds, have a number of drawbacks, including the need for manual selection to achieve a homogeneous population and the use of nonphysiological matrix compounds. In this study we describe a mild method for the generation of MCTS, in which individual spheroids form in hanging drops suspended from a microtiter plate. The method has been successfully applied to a broad range of cell lines and shows nearly 100% efficiency (i.e., one spheroid per drop). Using the hepatoma cell line, HepG2, the hanging drop method generated well-rounded MCTS with a narrow size distribution (coefficient of variation [CV] 10% to 15%, compared with 40% to 60% for growth on nonadherent surfaces). Structural analysis of HepG2 and a mammary gland adenocarcinoma cell line, MCF-7, composed spheroids, revealed highly organized, three-dimensional, tissue-like structures with an extensive extracellular matrix. The hanging drop method represents an attractive alternative for MCTS production, because it is mild, can be applied to a wide variety of cell lines, and can produce spheroids of a homogeneous size without the need for sieving or manual selection. The method has applications for basic studies of physiology and metabolism, tumor biology, toxicology, cellular organization, and the development of bioartificial tissue. (C) 2003 Wiley Periodicals, Inc.

Increased mononuclear cell activation and apoptosis early after human liver transplantation is associated with a reduced frequency of acute rejection

Experimental models of orthotopic liver transplantation (OLT) have shown that the very early events post-OLT are critical in distinguishing immunogenic and tolerogenic reactions. In rodents, increased leukocyte apoptosis and cytokine expression have been demonstrated in tolerogenic strain combinations. Information from human OLT recipients is less abundant. The aim of this study was to determine the amount of early leukocyte activation and apoptosis following human OLT, and to correlate this with subsequent rejection status. Peripheral blood mononuclear cells (PBMC) were isolated from 76 patients undergoing OLT - on the day prior, 5 hrs after reperfusion (day 0), and 18-24 hrs post-OLT (day 1). The mean level of apoptotic PBMCs on post OLT day 1 was higher than healthy recipients (0.9% +/- 0.2 vs. 0.2% +/- 0.1, p = 0.013). Apoptosis was greater in nonrejecting (NR) (1.1% +/- 0.3) compared with acutely-rejecting (R) (0.3% +/- 0.1, p = 0.021) patients. On day 1, PBMC from NR patients had increased expression of IFN-gamma (p = 0.006), IL-10 (p = 0.016), and CD40 ligand (p = 0.02) compared with R. Donor cell chimerism on day 1 did not differ between the groups indicating that this was unlikely to account for increased PBMC apoptosis in the NR group. Interestingly, the level of chimerism on day 0 was significantly higher in NR (3.8% +/- 0.6) compared with R (1.2% +/- 0.4, p = 0.004) patients and there was a close correlation between chimerism on day 0 and cytokine expression on day 1. These results imply that similar mechanisms are occurring in the human liver to promote graft acceptance as in the experimental models of liver transplantation and suggest that strategies that promote liver transplant acceptance in rodents might be applicable to humans.