Hyaluronan (HA) has traditionally been viewed as a structural component of the extracellular matrix, valued primarily for its viscoelastic, lubricating, and hydrating properties. However, accumulating evidence suggests that ultra-high molecular weight hyaluronan (UHMW-HA) functions far beyond passive tissue support, acting as a dynamic regulator of inflammation, mechanotransduction, immune homeostasis, cellular senescence, and regeneration. In this narrative review, we examine HA and specifically focus upon UHMW-HA operating as a “living hydrogel”, an adaptive regenerative macromolecule that continuously interacts with its microenvironment to shape tissue function and resilience. We examine evidence spanning evolutionary biology, glycobiology, biomaterials science, and regenerative medicine to examine how polymer size encodes biological activity and how UHMW-HA influences stem cell behaviour, extracellular matrix remodelling, and responses to injury and ageing. Attention is given to unique biological models, including the naked mole-rat, whose exceptional longevity and cancer resistance have been linked to abundant UHMW-HA, offering insights into the relationship between extracellular matrix architecture and organismal health. We further discuss how advances in synthetic biology and biomaterials engineering are enabling the development of next-generation HA-based hydrogels that mimic these adaptive properties for therapeutic applications in tissue engineering, in addition to potential roles in dynamically optimising wound repair, and musculoskeletal rejuvenation, through precision medicine approaches. Finally, we argue that reclassifying UHMW-HA as a living regenerative material rather than an inert scaffold provides a new concept for designing bioinspired therapeutics and understanding extracellular matrix function. This review demonstrates HA-adaptive macromolecules as critical entities in future regenerative strategies and highlights the specific and unique potential of UHMW-HA to combine material science developments with enhancement of preferable biological outcome.

Background: CD105 is highly expressed on human activated endothelial cells (ECs), is an important component of the TGF-β1 receptor complex and is essential for angiogenesis. CD105 expression is up-regulated in activated ECs and is an important potential marker for cancer prognosis. Materials and Methods: In vitro rat myoblasts transfected with the L-CD105 and S-CD105 transfectants. The transfectants were treated with TGF-β1 for the angiogenesis study. Results: L-CD105 affects cell proliferation in the presence and absence of TGF-β1, and inhibits p-ERK1/2, p-MEK1/2 and p-c-Jun in L-CD105 transfectants compared to controls. The induction of phospho-ERK1/2 following treatment with TGF-β1 remained significantly lower in L-CD105 transfectants compared to controls. Conclusion: L-CD105 inhibits the phosphorylation of ERK1/2, MEK1/2, c-Jun1/2/3, and associated signalling intermediates. CD105 modulates cell growth and TGF-β1 induced cell signalling through ERK-c-Jun expression.

Monomeric C-reactive protein (mCRP), the dissociated form of native C-reactive protein, is a critical molecule that causes and perpetuates inflammation in serious diseases. It has 'adhesive'-like properties causing aggregation of blood cells and platelets, and can stick permanently within arterial tissue where it can contribute to further complications including thrombosis, linking it potentially to atherosclerosis and subsequent acute coronary events. In this mini review, we discuss briefly the implications and the potential value of measuring and manipulating it for clinical diagnostics and therapeutic purposes.

Introduction: GRP94 is a 94-kDa glycoprotein abundant in the endoplasmic reticulum (ER) and is induced by glucose starvation.Overexpression of GRP94

The potential use of stem cells as therapeutics in disease has gained momentum over the last few years and recently phase-I clinical trials have shown favourable results in treatment of a small cohort of acute stroke patients. Similarly, they have been used in preclinical models drug-loaded for the effective treatment of solid tumours. Here we have characterized uptake and release of a novel p5-cyclin-dependent kinase 5 (CDK5) inhibitory peptide by mesenchymal stem cells and showed release levels capable of blocking aberrant cyclin-dependent kinase 5 (CDK5) signaling pathways, through phosphorylation of cyclin-dependent kinase 5 (CDK5) and p53. These pathways represent the major acute mechanism stimulating apoptosis after stroke and hence its modulation could benefit patient recovery. This work indicates a potential use for drug-loaded stem cells as delivery vehicles for stroke therapeutics and in addition as anticancer receptacles particularly, if a targeting and/or holding mechanism can be defined.

AIM to identify biological interactions between proliferating fibroblasts and HeLa cells in vitro. MATERIALS AND METHODS Fibroblasts were isolated from both normal and tumour human tissues. Coverslip co-cultures of HeLa and fibroblasts in various ratios with medium replacement every 48 h were studied using fixed cell staining with dyes such as Giemsa and silver staining, with immunochemistry for Ki-67 and E-cadherin, with dihydrofolate reductase (DHFR) enzyme reaction, as well as live cell staining for non-specific esterases and lipids. Other techniques included carmine cell labeling, autoradiography and apoptosis assessment. RESULTS Under conditions of feeding and cell: cell ratios allowing parallel growth of human fibroblasts and HeLa cells, co-cultured for up to 20 days, a series of phenomena occur consecutively: profound affinity between the two cell types and exchange of small molecules; encircling of the HeLa colonies by the fibroblasts and enhanced growth of both cell types at their contact areas; expression of carbonic anhydrase in both cell types and high expression of non-specific esterases and cytoplasmic argyrophilia in the surrounding fibroblasts; intense production and secretion of lipid droplets by the surrounding fibroblasts; development of a complex net of argyrophilic projections of the fibroblasts; E-cadherin expression in the HeLa cells; from the 10th day onwards, an increasing detachment of batches of HeLa cells at the peripheries of colonies and appearance of areas with many multi-nucleated and apoptotic HeLa cells, and small HeLa fragments; from the 17th day, appearance of fibroblasts blocked at the G2-M phase. Co-cultures at approximately 17-20 days display a cell-cell fight with foci of (a) sparse growth of both cell types, (b) overgrowth of the fibroblasts and (c) regrowth of HeLa in small colonies. These results indicate that during their interaction with HeLa cells in vitro, proliferating fibroblasts can be activated against HeLa. This type of activation is not observed if fibroblast proliferation is blocked by contact inhibition of growth at confluency, or by omitting replacement of the nutrient medium. CONCLUSION The present observations show that: (a) interaction between proliferating fibroblasts and HeLa cells in vitro drastically influences each other's protein expression, growth pattern, chromatin features and survival; (b) these functions depend on the fibroblast/HeLa ratio, cell topology (cell-cell contact and the architectural pattern developed during co-culture) and frequent medium change, as prerequisites for fibroblast proliferation; (c) this co-culture model is useful in the study of the complex processes within the tumour microenvironment, as well as the in vitro reproduction and display of several phenomena conventionally seen in tumour cytological sections, such as desmoplasia, apoptosis, nuclear abnormalities; and (d) overgrown fibroblasts adhering to the boundaries of HeLa colonies produce and secrete lipid droplets.

Transcription factor PAX3/Pax3 contributes to diverse cell lineages during embryonic development and is important in tumourigenesis. We found that PAX3 is re-expressed in neuroblastoma and malignant neuroblastic (N-type) neuroblastoma cells had significantly higher PAX3 protein expression than their benign substrate-adherent (S-type) counterparts. Knock-down of PAX3 expression by siRNA transfection resulted in persistent cell growth inhibition in both types of neuroblastoma cell, owing to G1 cell cycle arrest and progressive apoptosis. Inhibition of PAX3 expression significantly decreased the attachment of S-type SH-EP1 cells to extra-cellular matrix proteins, fibronectin, laminin and collagen IV. Migration and invasion of both neuroblastoma cell types were markedly reduced after PAX3 down-regulation. PAX3 knock-down significantly augmented the cytotoxic effect of chemotherapeutic agents, etoposide, vincristine and cisplatin, commonly used to treat neuroblastoma. Microarray analyses revealed that particularly signalling pathways involving cell cycle, apoptosis, cell adhesion, cytoskeletal remodelling and development were altered by PAX3 down-regulation. Changes in PAX3 downstream genes identified by microarray analyses were validated in 47 genes by quantitative PCR. These novel findings lead us to propose that PAX3 might contribute to oncogenic characteristics of neuroblastoma cells by regulating a variety of crucial signalling pathways.

Cyclin-dependent kinase-5 (Cdk5) is over-expressed in both neurons and microvessels in hypoxic regions of stroke tissue and has a significant pathological role following hyper-phosphorylation leading to calpain-induced cell death. Here, we have identified a critical role of Cdk5 in cytoskeleton/focal dynamics, wherein its activator, p35, redistributes along actin microfilaments of spreading cells co-localising with p((Tyr15))Cdk5, talin/integrin beta-1 at the lamellipodia in polarising cells. Cdk5 inhibition (roscovitine) resulted in actin-cytoskeleton disorganisation, prevention of protein co-localization and inhibition of movement. Cells expressing Cdk5 (D144N) kinase mutant, were unable to spread, migrate and form tube-like structures or sprouts, while Cdk5 wild-type over-expression showed enhanced motility and angiogenesis in vitro, which was maintained during hypoxia. Gene microarray studies demonstrated myocyte enhancer factor (MEF2C) as a substrate for Cdk5-mediated angiogenesis in vitro. MEF2C showed nuclear co-immunoprecipitation with Cdk5 and almost complete inhibition of differentiation and sprout formation following siRNA knock-down. In hypoxia, insertion of Cdk5/p25-inhibitory peptide (CIP) vector preserved and enhanced in vitro angiogenesis. These results demonstrate the existence of critical and complementary signalling pathways through Cdk5 and p35, and through which coordination is a required factor for successful angiogenesis in sustained hypoxic condition.

Cell-penetrating peptides (CPPs) have proven utility for the highly efficient intracellular delivery of bioactive cargoes that include peptides, proteins, and oligonucleotides. The many strategies developed to utilize CPPs solely as pharmacokinetic modifiers necessarily requires them to be relatively inert. Moreover, it is feasible to combine one or multiple CPPs with bioactive cargoes either by direct chemical conjugation or, more rarely, as non-covalent complexes. In terms of the message-address hypothesis, this combination of cargo (message) linked to a CPP (address) as a tandem construct conforms to the sychnological organization. More recently, we have introduced the term bioportide to describe monomeric CPPs that are intrinsically bioactive. Herein, we describe the design and biochemical properties of two rhegnylogically organized monometic CPPs that collectively modulate a variety of biological and pathophysiological phenomena. Thus, camptide, a cell-penetrant sequence located within the first intracellular loop of a human calcitonin receptor, regulates cAMP-dependent processes to modulate insulin secretion and viral infectivity. Nosangiotide, a bioportide derived from endothelial nitric oxide synthase, potently inhibits many aspects of the endothelial cell morphology and movement and displays potent anti-angiogenic activity in vivo. We conclude that, due to their capacity to translocate and target intracellular signaling events, bioportides represent an innovative generic class of bioactive agents.

Uncontrolled activation of calcium-sensitive calpain pathways induces neuronal damage during ischemic stroke. Vascular remodelling plays a critical role in tissue recovery. The main molecular events underpinning angiogenesis in stroke are still imprecise. Recently, we highlighted a novel non-neuronal role of the cyclin-dependent kinase (Cdk)-5 operating through p35 activator, which modulates in vitro brain angiogenesis. Cdk5 activators p35 and p25 were recently shown to be involved in neuronal protection (p35) or death (p25) after brain ischemia. However, the impact of p35/Cdk5 on angiogenesis during hypoxic injury remains unclear. Here, using the calpain inhibitor MDL28170 and GFP-Cdk5 wild type (wt) or kinase mutant (D144N) transfectants in an in vitro model of stroke, we tested the hypothesis that activation of p35/Cdk5 pathway may positively modulate angiogenesis during hypoxia. Human brain microvascular endothelial cells (hCMEC/D3) were treated (24h) with MDL28170 (10μM) under conditions of hypoxia (1%O 2 ) or normoxia. Angiogenesis was investigated by wound-healing assay and capillary-tube formation on matrigel, using Cell-IQ® Imaging system (Chip-Man Technologies Ltd). Cell viability was evaluated by assay of MTT proliferation, propidium iodide nuclear inclusion (nPI) and heat shock protein 70 (Hsp70) level. p35, Cdk5 and activated pY(15)Cdk5 intracellular localization were analyzed by confocal microscopy. Protein levels were estimated by western blot. Inhibition of calpain activity increased angiogenesis (p<0.01), augmenting p35 protein levels (30%). Hypoxia induced cell stress, as evidenced by increased Hsp70 (0.8 ±0.02 vs 0.5±0.01, ratio on GAPDH optical density) and nPIs, and hampered cell migration and tube formation (p<0.01). This was associated with reduced p35 (56%, ratio on GAPDH optical density) contents. Blocking Cdk5 activity with D144N transfectant reduced migration (p<0.05). Cdk5 (wt) overexpression enhanced migration, which was further augmented in hypoxia (p<0.05). Hypoxia impaired reorganization of the cytoskeleton and reduced p35/actin co-localization, without affecting the distribution of Cdk5 or pY(15)Cdk5 in association with actin filaments and focal tips. MDL28170 significantly reversed the effects of hypoxia on cell migration and p35 localization, suggesting a role of p35 in cytoskeleton stability. MDL28170 further increased cell migration in Cdk5(wt) or D144N either in hypoxia or normoxia, and Cdk5 protein levels in hypoxia (0.2 ±0.01 vs 0.18±0.01, ratio on GAPDH optical density), implying that increased p35 may be protective during stroke. In conclusion, our data suggest that p35/Cdk5 signaling supports angiogenesis in stroke. p35 may be a signalling intermediate, involved in cytoskeleton organization and cell dynamics during angiogenesis.

Background: Angiogenesis is important in health and several disease states. CD105 is a proliferation-associated and hypoxia-inducible transmembrane protein abundantly expressed in angiogenic endothelial cells. CD105 is a receptor for transforming growth factors (TGF)-‚1 and -‚3. The exact mechanisms for CD105 regulation of vascular development have not been fully elucidated. Materials and Methods: In this study, an antisense approach to create a murine and a human stably transfected endothelial cell line expressing a reduction in CD105 protein was used. Results: We showed that inhibition of CD105 in cultured murine and human endothelial cells enhanced the ability of TGF-‚1 to suppress growth and migration, and influenced TGF-‚1 promoter activity. TGF-‚1 not only reduced the length of the capillary-like structures, but also caused mortality in CD105-deficient murine antisense cells compared to control cultures. To determine whether CD105 affected TGF-‚1-induced gene expression, a luciferase assay in transiently transfected cells with p3TP-Lux promoter constructs was performed. Both murine and human antisense transfectants showed a significant increase in p3TP-Lux promoter activity. Further studies on the functional importance of CD105 was undertaken in irradiated normoxic and hypoxic cells. The levels of proand anti-apoptotic markers were also evaluated. There was an increase in pro-apoptotic marker (p53), but a reduction in anti-apoptotic marker (Bcl-2) in CD105-deficient cells. Conclusion: These results provide direct evidence that CD105 antagonises the inhibitory effects of TGF-‚1 on human and murine vascular endothelial cells and that normal cellular levels of CD105 are required for the formation of new blood vessels. CD105 (endoglin) is a 180kDa homodimeric integral membrane glycoprotein that is expressed primarily in the vascular endothelial cells of capillaries, arterioles and venules, as well as in activated monocytes, some leukemic cells and the syncytiotrophoblast, the multinucleated placental layer which constitutes the interface with maternal blood (1, 2). The pivotal role of CD105 in angiogenesis is demonstrated in knock-out mice, wherein targeted inactivation of the CD105 gene results in vascular and cardiovascular defects in early mouse embryos (3). From embryonic day 9, the primitive vascular plexus of the yolk sac fails to form mature structures leading to vessel dilation, rupture and haemorrhage (4, 5). CD105 binds both TGF-‚1 and TGF-‚3, is found in a signalling complex with TGF‚RI and TGF‚RII after ligand binding (6) and is an accessory protein for activin A and bone morphogenic protein 7 (7). The cytoplasmic domain of CD105 is 70% homologous to betaglycan (8). Studies using CD105 transformants revealed that CD105 overexpression prevented the inhibition of cellular proliferation and the down-regulation of c-myc mRNA induced by TGF-‚1 (9). TGF-‚1-induced stimulation of fibronectin synthesis, cellular adhesion, platelet-endothelial cell adhesion molecule-1 phosphorylation and homotypic aggregation were also inhibited in CD105-transfectants (6). In the absence of exogenous TGF-‚1, the synthesis of fibronectin and PAI-1 was reduced in mouse fibroblasts overexpressing CD105. In contrast, using an antisense approach, it was shown that the inhibition of CD105 expression in cultured endothelial cells enhanced the ability of TGF-‚1 to suppress their growth and migration (3). These data, and more recent publications, strongly suggest that CD105 interferes with specific cellular responses to TGF-‚1, and that it could modulate the intracellular signals transmitted by the TGF-‚ receptor complex, via the constitutive phosphorylation of the cytoplasmic domain and the formation of the heteromeric complex of CD105 with the TbRI/RII signal complex reviewed in Duff et al. (10). This is very important as down-regulation or loss of 1851 Correspondence to: Dr. M Chantal Hillarby, Division of Laboratory and Regenerative Medicine, First Floor, Stopford Building, University of Manchester, Oxford Road, Manchester, M13 9PT, U.K. Tel: 44 (0)161 275 5271, Fax: 44 (0)161 275 5272, e-mail: chantal.hillarby@manchester.ac.uk

The endothelium regulates vascular homeostasis and is responsible for angiogenesis, a process mediated by the sprouting of endothelial cells from pre-existing vessels. Several lines of evidence indicate that endothelial progenitor cells (EPCs) also play a role in adult neovascularization as well as in the maintenance of endothelial integrity and function. Hypercholesterolemia is associated with increased cardiovascular risk by inducing a cascade of events leading to endothelial dysfunction and injury. Growing evidence indicates that low-density lipoproteins (LDLs) impair endothelial reparative processes by inducing endothelial cell apoptosis but also by reducing the number and function of EPCs. The involvement of LDLs in mechanisms associated with vascular repair and neovascularization is also suggested by data from studies using lipid-lowering drugs (statins). This review is focused on the central role of the cholesterol pathway in the biology of the endothelium and EPCs.

The basement membrane, immune cells, capillaries, fibroblasts and extracellular matrix (ECM) constitute the tumour stroma, commonly referred to as the 'reactive stroma'. The fibroblasts from the initial stages of a tumour, as the main constituents of the reactive stroma, present a different phenotype from the normal fibroblasts and play a crucial role in tumour progression. This review presents the differences between normal and tumour stromal fibroblasts and analyzes the molecular mechanisms (which involve growth factors, ECM components, matrix metalloproteinases, integrins and cell adhesion molecules) in the complex interactions between stromal fibroblasts and tumour cells. To date, several examples of heterotypic interactions between tumour stromal fibroblasts and tumour cells have supported the hypothesis that the tumour stroma promotes the growth of the tumour mass, as well as invasion and metastasis. However, it remains possible that the stroma acts essentially as a local modulator to impede tumorigenesis at an early stage and that the desmoplastic response is a host defence reaction designed to confine the developing tumour. The latter hypothesis has largely been neglected. The review aims to give a broader view on the role of stromal fibroblasts in tumour growth, invasion and metastasis.