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.

Background Angiogenesis, the growth of new blood vessels from the pre-existing vasculature is associated with pathological processes, in particular tumour development, and is a target for the development of new therapies. We have investigated the anti-angiogenic potential of two naturally occurring stilbene glycosides (compounds 1 and 2 ) isolated from the medicinal plant Boswellia papyriferai using large and smallvessel-derived endothelial cells. Compound 1 (trans-4',5'-dihydroxy-3-methoxystilbene-5-O-{α-L-rhamnopyranosyl-(1→2)-[α-L-rhamnopyranosyl-(1→6)}-β-D-glucopyranoside was the more hydrophilic and inhibited FGF-2-induced proliferation, wound healing, invasion in Matrigel, tube formation and angiogenesis in large and small vessel-derived endothelial cells and also in the chick chorioallantoic membrane assay. Using a binding assay we were able to show compound 1 reduced binding of FGF-2 to fibroblast growth factor receptors-1 and -2. In all cases the concentration of compound 1 which caused 50% inhibition (IC 50 ) was determined. The effect of compound 1 on EGF and VEGF-induced proliferation was also investigated. Results Compound 1 inhibited all stages of FGF-2 induced angiogenesis with IC 50 values in the range 5.8 ± 0.18 – 48.90 ± 0.40 μM but did not inhibit EGF or VEGF-induced angiogenesis. It also inhibited FGF-2 binding to FGF receptor-1 and -2 with IC 50 values of 5.37 ± 1.04 and 9.32 ± 0.082 μM respectively and with concommotant down-regulation of phosphorylated-ERK-1/-2 expression. Compound 2 was an ineffective inhibitor of angiogenesis despite its structural homology to compound 1 . Conclusion Compound 1 inhibited FGF-2 induced angiogenesis by binding to its cognate receptors and is an addition to the small number of natural product inhibitors of angiogenesis

Background Formation of haemorrhagic neovessels in the intima of developing atherosclerotic plaques is thought to significantly contribute to plaque instability resulting in thrombosis. C-reactive protein (CRP) is an acute phase reactant whose expression in the vascular wall, in particular, in reactive plaque regions, and circulating levels increase in patients at high risk of cardiovascular events. Although CRP is known to induce a pro-inflammatory phenotype in endothelial cells (EC) a direct role on modulation of angiogenesis has not been established. Results Here, we show that CRP is a powerful inducer of angiogenesis in bovine aortic EC (BAEC) and human coronary artery EC (HCAEC). CRP, at concentrations corresponding to moderate/high risk (1–5 μg/ml), induced a significant increase in proliferation, migration and tube-like structure formation in vitro and stimulated blood vessel formation in the chick chorioallantoic membrane assay (CAM). CRP treated with detoxi-gel columns retained such effects. Western blotting showed that CRP increased activation of early response kinase-1/2 (ERK1/2), a key protein involved in EC mitogenesis. Furthermore, using TaqMan Low-density Arrays we identified key pro-angiogenic genes induced by CRP among them were vascular endothelial cell growth factor receptor-2 (VEGFR2/KDR), platelet-derived growth factor (PDGF-BB), notch family transcription factors (Notch1 and Notch3), cysteine-rich angiogenic inducer 61 (CYR61/CCN1) and inhibitor of DNA binding/differentiation-1 (ID1). Conclusion This data suggests a role for CRP in direct stimulation of angiogenesis and therefore may be a mediator of neovessel formation in the intima of vulnerable plaques.

Angiogenesis is a complex sequential process involving endothelial activation, basement membrane degradation, endothelial sprouting from the parent vessel, invasion of the extracellular matrix, endothelial proliferation, vessel elongation, branching, anastomosis, increases in vessel diameter, basement membrane formation, pericyte acquisition, and remodelling. Most in vitro angiogenesis assays are two-dimensional and measure only one facet of this process, generally endothelial proliferation, migration, or tube formation. The two-dimensional nature of the assays also ignores the differences in endothelial phenotype seen in three-dimensional models and in vivo. The in vitro serum-free three-dimensional rat aortic model closely approximates the complexities of angiogenesis in vivo, from endothelial activation to pericyte acquisition and remodelling, and most of these can be quantified by image analysis, immunohistochemistry, and biochemical analysis. It is easily manipulated using molecular biological intervention or exogenous inhibitors and activators in a relatively controlled system.

Angiogenesis, the growth of new blood vessels from the pre-existing vasculature is of physiological and pathological importance. We have investigated the anti-angiogenic potential of two naturally occurring sesterterpenes, leucosesterterpenone (compound 1) and leucosterlactone (compound 2) isolated from the Himalayan plant Leucosceptrum canum and identified as having biological activity in preliminary screening. Compound 1 inhibited fibroblast growth factor-2-induced proliferation, migration in a wounding assay, chemotaxis and tube formation with small vessel (human dermal) and large vessel (bovine aortic) endothelial cells while compound 2 was largely inactive. Both compounds were also active in an in vivo angiogenic model using the chick chorioallantoic membrane. Neither compounds showed inhibitory activity in the absence of fibroblast growth factor-2. We were able to demonstrate in a binding assay that compounds 1 and 2 bound to the fibroblast growth factor-2 receptor-1 with IC50 values of 1.4 +/- 0.956 and 132.47 +/- 7.90 mu M, respectively, with a concomitant down regulation of phosphorylated ERK1/2 but did not bind to receptor-2. Compound 1 was less hydrophobic than compound 2 and this may contribute to its increased activity. Compound 1 is a new addition to the small number of inhibitors of fibroblast growth factor-2-induced angiogenesis. The compound was a specific inhibitor in that it had no effect on vascular endothelial growth factor or epithelial growth factor-induced angiogenesis. Since angiogenesis is essential for tumour development we conclude that these compounds may have potential as antitumour agents.

Background Angiogenesis, the growth of new blood vessels from the pre-existing vasculature is associated with physiological (for example wound healing) and pathological conditions (tumour development). Vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2) and epidermal growth factor (EGF) are the major angiogenic regulators. We have identified a natural product (cheiradone) isolated from a Euphorbia species which inhibited in vivo and in vitro VEGF- stimulated angiogenesis but had no effect on FGF-2 or EGF activity. Two primary cultures, bovine aortic and human dermal endothelial cells were used in in vitro (proliferation, wound healing, invasion in Matrigel and tube formation) and in vivo (the chick chorioallantoic membrane) models of angiogenesis in the presence of growth factors and cheiradone. In all cases, the concentration of cheiradone which caused 50% inhibition (IC 50 ) was determined. The effect of cheiradone on the binding of growth factors to their receptors was also investigated. Results Cheiradone inhibited all stages of VEGF-induced angiogenesis with IC 50 values in the range 5.20–7.50 μM but did not inhibit FGF-2 or EGF-induced angiogenesis. It also inhibited VEGF binding to VEGF receptor-1 and 2 with IC 50 values of 2.9 and 0.61 μM respectively. Conclusion Cheiradone inhibited VEGF-induced angiogenesis by binding to VEGF receptors -1 and -2 and may be a useful investigative tool to study the specific contribution of VEGF to angiogenesis and may have therapeutic potential.

Angiogenesis plays a key role in ocular development and disease. Opticin is an extracellular matrix small leucine-rich repeat glycoprotein which was first isolated from vitreous humour of the eye. As it is highly expressed in the eye throughout life, we hypothesised that it may contribute towards the angiogenic process. The effect of opticin on FGF-2-stimulated angiogenesis was determined using assays for monolayer wound recovery and boyden chamber (migration), tube-like structure formation and invasion in matrigel or collagen gel, and examination of protein phosphorylation using Kinexus Western arrays (Bioinformatics, Canada). The presence of opticin decreased cell migration (31%, p< 0.005), tube like structure formation (37%, p<0.001) and cell invasion (45%, p<0.001). Opticin also significantly decreased the development of blood vessel formation in the chick chorioallantoic membrane assay. Opticin decreased FGF-2-induced phosphorylation of MEK1/2 (MAPK kinase), JNK and p38 (MAPK). A decrease of MAPKs phosphorylation is known to play crucial roles in cell migration and tubular morphogenesis. Therefore, opticin maybe an inhibitor of vascular ingression into the vitreous humour.

The correct formation of new blood vessels from existing vasculature (angiogenesis) is essential for embryogenesis and the effective repair of damaged or wounded tissues. However, excessive and detrimental vascularization also occurs in neoplasia, promoting tumour growth and metastasis, as well as in proliferative diabetic retinopathy and atherosclerosis. Greater understanding of the mechanisms controlling the angiogenic process will allow optimization of wound healing, and provide mechanisms to inhibit vascularization in tumours and other diseases. Evidence supports a cascade of events in which the perturbation of one of the steps is sufficient to significantly inhibit neovascularization. The extracellular macromolecules, notably glycosaminoglycans (GAGs), are important mediators of angiogenesis. Hyaluronan (HA), a large, non-sulphated GAG, was first discovered in the vitreous of the eye [Meyer, K., Palmer, J.W. 1936. The polysaccharide of the vitreous humour. J. Biol. Chem. 107, 629–634.], and is ubiquitously expressed in the extracellular matrix (ECM) of tissues. Native high molecular weight HA (n-HA) is anti-angiogenic, whereas HA degradation products (o-HA; 3–10 disaccharides) stimulate endothelial cell (EC) proliferation, migration and tube formation following activation of specific HA receptors in particular, CD44 and Receptor for HA-Mediated Motility (RHAMM, CD168). The involvement of HA in the regulation of angiogenesis makes it an attractive therapeutic target. We review the role of o-HA in modulation of angiogenesis during tissue injury, and vascular disease, focusing on receptor-mediated signal transduction pathways that have been evaluated.

The extent of recovery from stroke is dependent on the survival of neurons, particularly in peri-infarcted regions. Angiogenesis is critical for the development of new microvessels and leads to re-formation of collateral circulation, reperfusion and better recovery. Hyaluronan (HA) is an important component of the brain extracellular matrix and a regulator of cellular differentiation, migration, proliferation and angiogenesis. We have found that the production of total HA and low molecular mass 3-10 disaccharides of HA (o-HA) was increased in post-mortem tissue and in the serum of patients 1, 3, 7 and 14 days (peaking at 7 days) after ischaemic stroke. Hyaluronidase activity was also increased in serum samples (peaking after 3 days), which might explain the subsequent increase in o-HA. Affinity-histochemical staining was performed using a HA-specific biotinylated binding protein, and it showed enhanced deposition of HA in blood vessels and intracellularly as well as in the nuclei of peri-infarcted neurons. Western blotting and immunohistochemistry demonstrated upregulation of HA synthases (HAS1 and 2) and hyaluronidases (HYAL1 and 2) in inflammatory cells from both stroke and peri-infarcted regions of the brain. HYAL1 was upregulated in microvesssels and intracellularly in neurons, whilst HAS2 became translocated into the nuclei of neurons in peri-infarcted areas. Receptor for HA-mediated motility was observed intracellularly and in the nuclei of neurons, in the tunica media of larger blood vessels and in the endothelial cells of microvessels in stroke-affected tissue, whilst expression of other receptors for HA, CD44 and tumour necrosis factor-stimulated gene 6 (TSG-6) were mainly increased in infiltrating mononuclear cells from inflammatory regions. The data presented here demonstrate that HA breakdown is a feature of the acute stage of stroke injury. Increased o-HA production soon after stroke may be detrimental through enhancement of the inflammatory response, whilst activation of HA and/or o-HA-induced cellular signalling pathways in neurons and microvessels may impact on the remodelling process by stimulating angiogenesis and revascularization, as well as the survival of susceptible neurons.

BMRTC, bone-metastasising renal tumour of childhood; CAM, chorioallantoic membrane; EC, endothelial cell; ECM, extracellular matrix; HA, hyaluronan; HAase, hyaluronidase; nHA, native HA; o-HA, oligosaccharides of HA. Dear Sir, We congratulate Dr. Lokeshwar's group for another landmark paper in this journal, wherein they demonstrated that the saliva of patients with high-grade head-and-neck squamous cell carcinoma contained both high and low m.w. HA.1 The latter, i.e., o-HA, is generated when HAase, an endoglycosidase, degrades HA. Although the authors did allude to the issue of HA and its relevance to angiogenesis, we highlight here its pathologic relevance in human malignancy and present our supporting unpublished data. The angiogenic activity of HA is dependent on its molecular mass. High m.w. nHA is antiangiogenic, whereas almost 2 decades ago we reported that HA degradation products of a specific size (3–25 disaccharide units) induce angiogenesis in vivo.2 Subsequently, we have shown that o-HA fragments of 3–10 disaccharides were angiogenic in several in vivo assays and stimulated EC migration, proliferation and sprout formation.3, 4, 5, 6 The biological activities of HA are mediated through interaction with cell surface receptors such as CD44, resulting in activation of intracellular signalling events. In collaboration with Dr. R. Montesano's group, we found that HA degradation products influence EC invasion of a 3-D ECM.7 o-HA acted synergistically with vascular endothelial growth factor but not basic fibroblast growth factor. We reported that o-HA induces phosphorylation and activation of mitogen-activated protein kinase in bovine ECs, as well as upregulating the early response genes c-fos, c-jun and jun-B, which control the expression of other genes, including those of matrix-degrading proteases.8 Because HA acts via its receptor, CD44, it is conceivable that o-HA promotes cell invasion and tube formation by activating intracellular signalling pathways that result in modulation of pericellular proteolysis. Also, o-HA induced multiple signalling pathways involved in EC migration, proliferation and wound healing.9, 10 More relevant to Lokeshwar's publication is our finding that sera of children with renal tumours contain o-HA. Briefly, renal tumours are one of the major groups of childhood solid malignancy,Wilms' tumour (nephroblastoma) being the most frequent in this group. Another kidney tumour BMRTC has been distinguished from Wilms' tumour.11 BMRTC comprises about 4% of all primary renal neoplasms in childhood. In the Manchester region, with a population of 1 million children, we expect to encounter approximately one BMRTC case every 5 years. BMRTC has been associated with poor prognosis, unlike Wilms' tumour.11 Furthermore, in contrast to Wilms' tumour, BMRTC occurs predominantly in boys and, as the name implies, frequently metastasises to bone (in 60% of cases compared to 1% for Wilms' tumour). HA is a normal component of human serum. Its half-life in the circulation is 2.5–5.5 min and its mean serum concentration, 42 ± 25 μg/l in healthy adults.12 In contrast to patients with Wilms' tumour and mesothelioma, patients with many other types of cancer have serum HA levels within the normal range.13, 14 It is possible that the m.w. of HA, rather than its level in serum, may be pathognomonic for certain cancers. Indeed, we found that the HA level in the sera of normal children was barely detectable and had a m.w. of 1 to 5 x 10.5 In both Wilms' and BMRTC patients, very high levels of HA were found in preoperative serum samples; these fell markedly following surgical excision of tumours.15 We also demonstrated the presence of low m.w. HA (similar to the angiogenic fragment of HA) in the sera of BMRTC patients. In contrast, high m.w. HA (which is not angiogenic) was found in the sera of Wilms' patients. Following surgery in BMRTC patients, not only did serum HA levels fall to a value within normal ranges but also the HA which remained was of high m.w. We now have unpublished data to show that tissue culture medium from BMRTC cells, in contrast to Wilms' tumours, contains low m.w. o-HA. Furthermore, FPLC separation of HAase-digested n-HA (kindly provided by Dr. I. Scott, Unilever, London, UK) has enabled us to narrow down the HA size to between 5 and 7 disaccharides, which continue to retain their angiogenic potential. None of these disaccharides individually was able to induce angiogenesis in chicken CAM assay. However, in combination, they had the ability to induce angiogenesis in vivo and EC migration in vitro. Future studies should be directed toward ascertaining whether the size of o-HA is a hallmark of a certain type of malignancy. Yours sincerely, Mark Slevin, David West, Pat Kumar, Paul Rooney, Shant Kumar

Angiogenesis plays a key role in ocular development and disease. Normally following regression of the hyaloid vascular system the vitreous humour is avascular and anti-angiogenic. However, in diseases such as proliferative diabetic retinopathy, new blood vessels grow from the retina into the vitreous humour potentially leading to blindness. Opticin is a glycoprotein that was first isolated from vitreous humour of the eye and is a member of the extracellular matrix small leucine-rich repeat proteoglycan/protein family. As it is highly expressed in the eye throughout life we hypothesised that it contributes towards the anti-angiogenic properties of the vitreous humour. Purified recombinant opticin was generated as previously described (Le Goff et al. JBC 2003, 278:45280). Using the recombinant opticin we show that opticin potently inhibits VEGF and FGF-2-induced human retinal endothelial cell and bovine aortic endothelial cell proliferation, migration, formation of tube-like structures in matrigel and collagen, and invasion. In vivo studies showed that opticin significantly decreased FGF-2 and VEGF induced angiogenesis in chick chorioallantoic membrane assays. We also demonstrate that opticin decreases signalling through ERK1/2. These observations suggest that opticin is a key regulator of angiogenesis in the eye and further studies will elucidate its mechanisms of action.

The study of the angiogenic process and the search for novel therapeutic agents to inhibit, or stimulate, angiogenesis has employed a wide range of in vivo 'angiogenesis' assays (reviewed in 1-3). These differ greatly in their difficulty, quantitative nature, rapidity, and cost. The classical in vivo models include the rabbit ear chamber, hamster cheek pouch, dorsal skin chamber, dorsal skin and air-sac model, anterior chamber/iris and avascular corneal pocket assay, and the chick embryo chorioallantoic membrane (CAM) assay. More recent methods involve the implantation of preloaded Matrigel or alginate plugs, or collagen or poly vinyl sponges (1). Largely owing to its simplicity and low cost, the CAM is the most widely used in vivo model for the study of both angiogenesis and antiangiogenesis (1,4).

ABSTRACT High-molecular-weight hyaluronan (HA) is inhibitory in both in vivo and in vitro models of angiogenesis. However, a discrete fraction of HA-degradation products have consistently been found stimulate angiogenesis in these same models. Recent studies in wound healing models revealed a close temporal relationship between tissue hyaluronidase activity, tissue HA-degradation and neovascularization. It appears that HA-degradation is a prerequisite for the induction of wound angiogenesis. On going studies, with transplantable tumours and tumour cell-lines, indicate that both metastatic properties and angiogenesis are associated with elevated levels of hyaluronidase and HA degradation. RT-PCR analysis suggests that this is due to an aberrant, or elevated, expression of the GPI-anchored cell-surface hyaluronidase PH-20 and, in some cell-lines, the HYAL1 hyaluronidase. PH-20 expression increases with both the angiogenic and metastatic potential of these cell-lines i.e. tumour progression. The role of HYAL1 is more controversial, but the present study suggests that in some cell-lines this may also play a role in the progression of tumours to a metastatic/angiogenic phenotype.

For many years the idea of the "magic bullet" approach to target cells in the treatment of cancer and other pathological conditions has been a tempting scenario. One such approach is to develop monoclonal antibodies (MAbs) against defined cell-surface markers and use these antibodies to direct cytotoxic agents to specific cells. However, the use of such therapies to target the actual tumor cells within solid tumors has proved to be inefficient, with usually less than 0.01% of the injected dose of an antibody localising per gram of tumor in humans. Furthermore, owing to the density of the packed tumor cells and high interstitial pressure in the tumor core, this localization is uneven and the antibody tends to become adsorbed in the perivascular regions of the peripheral tumor cells, with none reaching the tumor cells at more distant sites.

Macromolecular hyaluronan (HA) has consistently been shown to inhibit angiogenesis, in both in vivo and in vitro experimental models. The inhibitory effects of HA appear to be dependent on its size and concentration. Examination of the relationship between HA metabolism and vascularization in wound healing models has shown a close temporal coincidence between HA-degradation and the onset and rate of neovascularization. As in previous developmental studies, an increase in tissue hyaluronidase activity accompanied the degradation of tissue HA and angiogenesis. Recent studies on transplantable tumors and cultured tumor cell lines indicate that tumor metastasis and angiogenesis are associated with increased HA degradation and elevated hyaluronidase levels. These data suggest that the onset and degree of tissue angiogenesis is dependent on the level of hyaluronidase-mediated degradation of tissue HA. At least in tumors, this appears to be due to a GPI-anchored "neutral" cell surface hyaluronidase similar to the sperm surface hyaluronidase, PH-20. RT-PCR and Northern analysis confirmed the presence of PH-20 in most tumor cell lines. The level of PH-20 expression increased with the level of angiogenesis and metastatic potential. Using substrate gel electrophoresis and PCR, a second extracellular hyaluronidase, HYAL1, was detected in some cell lines, but its significance is not yet clear.

The microenvironment of the majority of solid tumours in which new vessels must grow and survive is acidic. Whilst recent reports suggest a role of the low tumour pH in the invasive and metastatic potential of tumour cells, little is known as to its impact on angiogenesis. The three-dimensional in vitro rat aortic ring model was used to study the effects of low extracellular pH (pH e ) on microvascular growth. The spontaneous angiogenic response in collagen gels was seen to be highly dependent on the pH of the culture medium, with optimal outgrowth at pH 7.4, and a marked delay in microvascular growth at pH 6.9. This inhibition of vascular development was reversible. The absence of similar effects of medium pH on monolayer outgrowths of endothelial cells from rat aortic rings suggested an effect of pH e on aspects specific to three-dimensional growth. Vascular endothelial growth factor and basic fibroblast growth factor, whilst having limited effects at pH 7.4, were seen to reduce the time to onset of vessel outgrowth at pH 7.1, and lead to an initial growth rate similar to that observed at pH 7.4 in the absence of growth factors. Thus, the low environmental pH encountered by endothelial cells in solid tumours would not necessarily be detrimental to neovascularisation: a prominent in vitro angiogenic response is still observed at low pH e when stimulated by exogenous growth factors, high concentrations of which would be present in vivo .