Multimerin-2 is a ligand for group 14 family C-type lectins CLEC14A, CD93 and CD248 spanning the endothelial pericyte interface.

The C-type lectin domain containing group 14 family members CLEC14A and CD93 are proteins expressed by endothelium and are implicated in tumour angiogenesis. CD248 (alternatively known as endosialin or tumour endothelial marker-1) is also a member of this family and is expressed by tumour-associated fibroblasts and pericytes. Multimerin-2 (MMRN2) is a unique endothelial specific extracellular matrix protein that has been implicated in angiogenesis and tumour progression. We show that the group 14 C-type lectins CLEC14A, CD93 and CD248 directly bind to MMRN2 and only thrombomodulin of the family does not. Binding to MMRN2 is dependent on a predicted long-loop region in the C-type lectin domain and is abrogated by mutation within the domain. CLEC14A and CD93 bind to the same non-glycosylated coiled-coil region of MMRN2, but the binding of CD248 occurs on a distinct non-competing region. CLEC14A and CD248 can bind MMRN2 simultaneously and this occurs at the interface between endothelium and pericytes in human pancreatic cancer. A recombinant peptide of MMRN2 spanning the CLEC14A and CD93 binding region blocks CLEC14A extracellular domain binding to the endothelial cell surface as well as increasing adherence of human umbilical vein endothelial cells to the active peptide. This MMRN2 peptide is anti-angiogenic in vitro and reduces tumour growth in mouse models. These findings identify novel protein interactions involving CLEC14A, CD93 and CD248 with MMRN2 as targetable components of vessel formation.

Proline-Rich Homeodomain protein (PRH/HHEX) is a suppressor of breast tumour growth.

Breast tumours progress from hyperplasia to ductal carcinoma in situ (DCIS) and invasive breast carcinoma (IBC). PRH/HHEX (proline-rich homeodomain/haematopoietically expressed homeobox) is a transcription factor that displays both tumour suppressor and oncogenic activity in different disease contexts; however, the role of PRH in breast cancer is poorly understood. Here we show that nuclear localization of the PRH protein is decreased in DCIS and IBC compared with normal breast. Our previous work has shown that PRH phosphorylation by protein kinase CK2 prevents PRH from binding to DNA and regulating the transcription of multiple genes encoding growth factors and growth factor receptors. Here we show that transcriptionally inactive phosphorylated PRH is elevated in DCIS and IBC compared with normal breast. To determine the consequences of PRH loss of function in breast cancer cells, we generated inducible PRH depletion in MCF-7 cells. We show that PRH depletion results in increased MCF-7 cell proliferation in part at least due to increased vascular endothelial growth factor signalling. Moreover, we demonstrate that PRH depletion increases the formation of breast cancer cells with cancer stem cell-like properties. Finally, and in keeping with these findings, we show that PRH overexpression inhibits the growth of mammary tumours in mice. Collectively, these data indicate that PRH plays a tumour suppressive role in the breast and they provide an explanation for the finding that low PRH mRNA levels are associated with a poor prognosis in breast cancer.

Blocking CLEC14A-MMRN2 binding inhibits sprouting angiogenesis and tumour growth.

We previously identified CLEC14A as a tumour endothelial marker. Here we show that CLEC14A is a regulator of sprouting angiogenesis in vitro and in vivo. Using a human umbilical vein endothelial cell spheroid-sprouting assay, we found CLEC14A to be a regulator of sprout initiation. Analysis of endothelial sprouting in aortic ring and in vivo subcutaneous sponge assays from clec14a(+/+) and clec14a(-/-) mice revealed defects in sprouting angiogenesis in CLEC14A-deficient animals. Tumour growth was retarded and vascularity reduced in clec14a(-/-) mice. Pull-down and co-immunoprecipitation experiments confirmed that MMRN2 binds to the extracellular region of CLEC14A. The CLEC14A-MMRN2 interaction was interrogated using mouse monoclonal antibodies. Monoclonal antibodies were screened for their ability to block this interaction. Clone C4, but not C2, blocked CLEC14A-MMRN2 binding. C4 antibody perturbed tube formation and endothelial sprouting in vitro and in vivo, with a similar phenotype to loss of CLEC14A. Significantly, tumour growth was impaired in C4-treated animals and vascular density was also reduced in the C4-treated group. We conclude that CLEC14A-MMRN2 binding has a role in inducing sprouting angiogenesis during tumour growth, which has the potential to be manipulated in future antiangiogenic therapy design.

The effects of CCR5 inhibition on regulatory T-cell recruitment to colorectal cancer.

BACKGROUND: Regulatory T cells (Treg) are enriched in human colorectal cancer (CRC) where they suppress anti-tumour immunity. The chemokine receptor CCR5 has been implicated in the recruitment of Treg from blood into CRC and tumour growth is delayed in CCR5-/- mice, associated with reduced tumour Treg infiltration. METHODS: Tissue and blood samples were obtained from patients undergoing resection of CRC. Tumour-infiltrating lymphocytes were phenotyped for chemokine receptors using flow cytometry. The presence of tissue chemokines was assessed. Standard chemotaxis and suppression assays were performed and the effects of CCR5 blockade were tested in murine tumour models. RESULTS: Functional CCR5 was highly expressed by human CRC infiltrating Treg and CCR5(high) Treg were more suppressive than their CCR5(low) Treg counterparts. Human CRC-Treg were more proliferative and activated than other T cells suggesting that local proliferation could provide an alternative explanation for the observed tumour Treg enrichment. Pharmacological inhibition of CCR5 failed to reduce tumour Treg infiltration in murine tumour models although it did result in delayed tumour growth. CONCLUSIONS: CCR5 inhibition does not mediate anti-tumour effects as a consequence of inhibiting Treg recruitment. Other mechanisms must be found to explain this effect. This has important implications for anti-CCR5 therapy in CRC.

Identification of novel vascular targets in lung cancer.

BACKGROUND: Lung cancer remains the leading cause of cancer-related death, largely owing to the lack of effective treatments. A tumour vascular targeting strategy presents an attractive alternative; however, the molecular signature of the vasculature in lung cancer is poorly explored. This work aimed to identify novel tumour vascular targets in lung cancer. METHODS: Enzymatic digestion of fresh tissue followed by endothelial capture with Ulex lectin-coated magnetic beads was used to isolate the endothelium from fresh tumour specimens of lung cancer patients. Endothelial isolates from the healthy and tumour lung tissue were subjected to whole human genome expression profiling using microarray technology. RESULTS: Bioinformatics analysis identified tumour endothelial expression of angiogenic factors, matrix metalloproteases and cell-surface transmembrane proteins. Predicted novel tumour vascular targets were verified by RNA-seq, quantitative real-time PCR analysis and immunohistochemistry. Further detailed expression profiling of STEAP1 on 82 lung cancer patients confirmed STEAP1 as a novel target in the tumour vasculature. Functional analysis of STEAP1 using siRNA silencing implicates a role in endothelial cell migration and tube formation. CONCLUSIONS: The identification of cell-surface tumour endothelial markers in lung is of interest in therapeutic antibody and vaccine development.

Differential cavitation, angiogenesis and wound-healing responses in injured mouse and rat spinal cords.

The vascular disruption, blood vessel loss and cavitation that occur at spinal cord injury (SCI) epicenters in mice and rats are different, but few studies have compared the acute SCI response in the two species. This is of interest since key elements of the rat SCI response are shared with humans. In this study, we investigated acute SCI responses and characterized changes in pro- and anti-angiogenic factors and matrix deposition in both species. Cavitation was absent in mouse but the area of the lesion site was 21- and 27-fold larger at 8 and 15 days post-lesion (dpl), respectively, in the rat compared to intact control. The absence of wound cavitation in the mouse was correlated with increased levels of immunoreactive pro-angiogenic, pro-matrix and pro-wound-healing factors, e.g. laminin, matrix metalloproteinase-1 (MMP-1) and vascular endothelial growth factor-A (VEGF-A) within the wound, which were 6.0-, 2.9-, and 2.8-fold, respectively, higher in the mouse compared to rats at 8 dpl. Increased axonal sparing was observed after dorsal column (DC) injury, detected by higher levels of neurofilament 200 (NF200) immunoreactivity in the dorsal column of mice compared to rats at both T7 and T9 spinal segments. Despite similar post SCI deficits in plantar heat tests at 2h after injury (1.4- and 1.6-fold lower than control mice and rats, respectively), by 7 days the magnitude of these responses were comparable to sham-treated controls in both species, while no post-SCI changes in Von Frey hair filament test response were observed in either species. We conclude that the more robust angiogenesis/wound-healing response in the mouse attenuates post-injury wound cavitation. Although the spinal cord functions that were monitored post-injury were similarly affected in both species, we suggest that the quality of the angiogenesis/wound-healing response together with the diminished lesion size seen after mouse SCI may protect against secondary axon damage and create an environment more conducive to axon sprouting/regeneration. These results suggest the potential therapeutic utility of manipulating the angiogenic response after human SCI.

Analyzing shoulder translation with navigation technology.

PURPOSE: Asymmetric stress imposed on the shoulder can lead to anterior shoulder instability in young athletes who perform repetitive overhead motions. A common treatment, surgical anterior capsule tightening, assumes that the instability is caused by abnormal anterior laxity. This study investigated the possibility that one element of overall imbalance, posterior capsular tightness, could be an underlying reason for shoulder instability. Surgical navigation technology, which is more accurate than whole-body motion-capture systems, was used to study anterior translational motions. METHOD: The study was used four cadaver shoulders, with the scapula and rotator cuff muscles intact. Opto-electronic surgical navigation localization devices were mounted on the scapula and humerus to accurately capture positions and orientations. The shoulders were passively moved through 7 motions, 5 of simple angulation and 2 combinations of clinical interest. Each motion was repeated in 4 different soft-tissue states: rotator cuff intact, capsule intact, and surgically induced capsular tightnesses of 5 and 10mm. RESULTS: The shoulders had significantly greater anterior translation when the posterior capsule was artificially tightened (p < 0.05); this was particularly in movements that combined abduction with internal or external rotation, which are typical overhead sports motions. Overall translation was indifferent to whether the shoulders were intact or dissected down to the capsule, as was translation during flexion was indifferent to dissection state (p > 0.95). CONCLUSION: Surgical navigation technology can easily be used to analyze cadaveric shoulder motion, with opportunities for adaptation to anesthetized patients. Results suggest that the inverse of artificial tightening, such as surgical release of the posterior capsule, may be an effective minimally invasive treatment of chronic shoulder dislocation subsequent to sports motions.

Identification and angiogenic role of the novel tumor endothelial marker CLEC14A.

Tumor endothelial markers (TEMs) that are highly expressed in human tumor vasculature compared with vasculature in normal tissue hold clear therapeutic potential. We report that the C-type lectin CLEC14A is a novel TEM. Immunohistochemical and immunofluorescence staining of tissue arrays has shown that CLEC14A is strongly expressed in tumor vasculature when compared with vessels in normal tissue. CLEC14A overexpression in tumor vessels was seen in a wide range of solid tumor types. Functional studies showed that CLEC14A induces filopodia and facilitates endothelial migration, tube formation and vascular development in zebrafish that is, CLEC14A regulates pro-angiogenic phenotypes. CLEC14A antisera inhibited cell migration and tube formation, suggesting that anti-CLEC14A antibodies may have anti-angiogenic activity. Finally, in endothelial cultures, expression of CLEC14A increased at low shear stress, and we hypothesize that low shear stress due to poor blood flow in the disorganized tumor vasculature induces expression of CLEC14A on tumor vessels and pro-angiogenic phenotypes.

Induction of thrombospondin-1 partially mediates the anti-angiogenic activity of dexrazoxane.

BACKGROUND: Considerable interest lies in the identification of novel anti-angiogenic compounds for cancer therapy. We have investigated whether dexrazoxane has anti-angiogenic properties and if so, the mechanism of the inhibition. METHODS: The phenotypic effects of dexrazoxane on endothelial cell behaviour was investigated both in vitro using human umbilical vein endothelial cells (HUVECs) in cell proliferation, migration, cell cycle and aortic ring assays; and in vivo using the mouse angiogenesis subcutaneous sponge assay. Custom angiogenesis pathway microarrays were used to identify differentially expressed genes in endothelial cells after treatment with dexrazoxane vs a control. The differentially expressed genes were validated using real-time RT-PCR and western blotting; and the functional effect of one induced gene was confirmed using siRNA technology. RESULTS: Treatment of endothelial cells with dexrazoxane resulted in a dose-response inhibition of cell growth lasting for up to 5 days after a single dose of the drug. Dexrazoxane was inhibitory in the aortic ring tube forming assay and strongly anti-angiogenic in vivo in the rodent subcutaneous sponge model. The anti-angiogenic effect in the sponge was seen after systemic injection into the tail vein as well as after direct injection of dexrazoxane into the sponge. Treatment of microvascular endothelial cells in vitro with subtoxic doses of dexrazoxane stimulated thrombospondin-1 (THBS-1) secretion. Knockdown of THBS-1 with siRNA removed the angiogenesis inhibition effect of dexrazoxane, which is consistent with the anti-angiogenic and vascular normalising properties of the drug being principally mediated by THBS-1. CONCLUSION: We show that dexrazoxane administered in small repeated doses is strongly anti-angiogenic and that this activity is mediated by induction of the anti-angiogenic THBS-1 in endothelial cells.

Humeral head translation during glenohumeral abduction following computer-assisted shoulder hemiarthroplasty.

This study compared the effect of a computer-assisted and a traditional surgical technique on the kinematics of the glenohumeral joint during passive abduction after hemiarthroplasty of the shoulder for the treatment of fractures. We used seven pairs of fresh-frozen cadaver shoulders to create simulated four-part fractures of the proximal humerus, which were then reconstructed with hemiarthroplasty and reattachment of the tuberosities. The specimens were randomised, so that one from each pair was repaired using the computer-assisted technique, whereas a traditional hemiarthroplasty without navigation was performed in the contralateral shoulder. Kinematic data were obtained using an electromagnetic tracking device. The traditional technique resulted in posterior and inferior translation of the humeral head. No statistical differences were observed before or after computer-assisted surgery. Although it requires further improvement, the computer-assisted approach appears to allow glenohumeral kinematics to more closely replicate those of the native joint, potentially improving the function of the shoulder and extending the longevity of the prosthesis.

Tumor stroma as a target in cancer.

Solid tumors are composed of the malignant cell itself (most commonly a carcinoma) and supporting cells that comprise the stroma. Significant stromal components include the extracellular matrix, supporting fibroblasts, vessels comprised of endothelium, pericytes and in some cases vascular smooth muscle, lymphatics and usually a major leukocyte infiltration. Indeed, macrophages may constitute up to 50% of the viable cells within the tumor. For many years, researchers have concentrated almost exclusively on the malignant carcinoma and looked for ways to either selectively kill or restrict its growth. In recent years the frustrating lack of advances in cytotoxic cancer therapy provoked a search for more novel strategies and foremost amongst these were anti-angiogenesis and vascular targeting. The purpose of this article is to illustrate how the stroma is now being pursued as an anti-cancer target. The article will briefly touch on anti-angiogenics that are now entering the clinic but concentrate on recent studies looking at vascular disrupting agents, stromal tumor fibroblasts and macrophages. Target identification is illustrated by the search for tumor endothelial markers. Finally, we draw attention to efforts to develop a cancer vaccine. The genetic instability and variation found in carcinoma cells made vaccination in the past a near impossibility. In contrast, genetically stable tumor endothelium with its unique accessibility to blood borne agents, together with recent advances in immunotherapy means that the possibility of a cancer vaccine now takes on a reality not previously recognised.

CT scan method accurately assesses humeral head retroversion.

Humeral head retroversion is not well described with the literature controversial regarding accuracy of measurement methods and ranges of normal values. We therefore determined normal humeral head retroversion and assessed the measurement methods. We measured retroversion in 65 cadaveric humeri, including 52 paired specimens, using four methods: radiographic, computed tomography (CT) scan, computer-assisted, and direct methods. We also assessed the distance between the humeral head central axis and the bicipital groove. CT scan methods accurately measure humeral head retroversion, while radiographic methods do not. The retroversion with respect to the transepicondylar axis was 17.9 degrees and 21.5 degrees with respect to the trochlear tangent axis. The difference between the right and left humeri was 8.9 degrees. The distance between the central axis of the humeral head and the bicipital groove was 7.0 mm and was consistent between right and left humeri. Humeral head retroversion may be most accurately obtained using the patient's own anatomic landmarks or, if not, identifiable retroversion as measured by those landmarks on contralateral side or the bicipital groove.

Expression of pheromone receptor gene families during olfactory development in the mouse: expression of a V1 receptor in the main olfactory epithelium.

In the mouse, two large gene families, V1R and V2R, encoding putative pheromone receptors have been described. Studies have suggested a homotypic recognition role for V1Rs and V2Rs during development in the targeting of vomeronasal axons to specific sets of glomeruli in the accessory olfactory bulb (AOB). Analysis of the onset of expression of the V1R and V2R gene families in developing vomeronasal neurons using polymerase chain reaction and in situ hybridization now suggests that a role for these receptors in the organization of axon projections is only likely at the final stages of targeting within the AOB. Surprisingly, our studies reveal expression of a V1Rd receptor in scattered cells within the main olfactory epithelium, suggesting that limited pheromone detection may also take place in this structure. The pheromone sensory neurons of the vomeronasal system and the neuroendocrine gonadotrophin-releasing hormone (GnRH) neurons that regulate fertility both arise from progenitor cells of the nasal placode. The development of these two cell types is intimately linked, and the GnRH neuron population migrates into the forebrain during embryogenesis in close association with a subset of vomeronasal sensory axons; how GnRH neurons recognize this axon subset is unknown. We report selective expression of a V1Ra gene in the clonal NLT GnRH cell line, raising the possibility of a similar role for V1Rs or V2Rs in the directed migration of GnRH neurons. However, no expression of this gene or of other V1Rs and V2Rs is detectable at the cellular level in migrating GnRH neurons in the mouse.

Adrenomedullin and tumour angiogenesis.

The angiogenic activity of peptide adrenomedullin (AM) was first shown in 1998 . Since then, a number of reports have confirmed the ability of AM to induce the growth and migration of isolated vascular endothelial and smooth muscle cells in vitro and to promote angiogenesis in xenografted tumours in vivo. In addition, knockout murine models point to an essential role for AM in embryonic vasculogenesis and ischaemic revascularisation. AM expression is upregulated by hypoxia (a typical feature of solid tumours) and a potential role as a regulator of carcinogenesis and tumour progression has been proposed based on studies in vitro and in animal models. Nevertheless, translational research on AM, and in particular, confirmation of its importance in the vascularisation of human tumours has lagged behind. In this commentary, we review current progress and potential directions for future research into the role of AM in tumour angiogenesis.

The realisation of targeted antitumour therapy.

Better understanding of the pathways regulating proliferation and metastasis of cancer cells has led to the development of novel molecular-targeted therapies. The number of molecular-targeted agents approved for use in the clinic is growing, with many more in clinical trials. Most of these compounds can be broadly classified into two main categories: monoclonal antibodies and small-molecule tyrosine kinase inhibitors. The pathological processes targeted include vascular endothelial growth factor-dependent tumour angiogenesis and epidermal growth factor receptor-dependent tumour cell proliferation and survival. Unlike conventional chemotherapy, molecular-targeted agents offer the potential advantages of a relatively high therapeutic window and use in combination with other anticancer strategies without overlapping toxicity. It is hoped that these drugs will become valuable therapeutic tools within the multimodal approach to treating cancer. Recent progress in targeted antitumour therapy is discussed, with a focus on antiangiogenesis.

Cooperative stimulation of vascular endothelial growth factor expression by hypoxia and reactive oxygen species: the effect of targeting vascular endothelial growth factor and oxidative stress in an orthotopic xenograft model of bladder carcinoma.

Elevated thymidine phosphorylase has been shown to correlate with increased angiogenesis and poor prognosis in many cancers including transitional cell carcinoma of the bladder. In vitro studies have demonstrated that thymidine phosphorylase activity causes cellular oxidative stress and increases secretion of vascular endothelial growth factor. In this study, we show that thymidine phosphorylase activity also augments levels of the hypoxia-inducible factor-1alpha during in vitro hypoxia, and that thymidine phosphorylase activity and hypoxia act in concert to increase vascular endothelial growth factor (VEGF) secretion. We also demonstrate that thymidine phosphorylase overexpression confers tumorigenicity on an orthotopically implanted transitional cell carcinoma cell line. Administration of the antioxidant N-acetylcysteine together with a blocking anti-VEGF antibody abrogates the increase in tumorigenicity. Our results support the increased efficacy of combination approaches to antiangiogenic therapy.

Disruption of ephrin signaling associates with disordered axophilic migration of the gonadotropin-releasing hormone neurons.

Ephrin signaling is involved in repulsive and attractive interactions mediating axon guidance and cell-boundary formation in the developing nervous system. As a result of a fortuitous transgene integration event, we have identified here a potential role for EphA5 in the axophilic migration of gonadotropin-releasing hormone (GnRH) neurons from the nasal placode into the brain along ephrin-expressing vomeronasal axons. Transgene integration in the GNR23 mouse line resulted in a 26 kb deletion in chromosome 5, approximately 67 kb 3' to Epha5. This induced a profound, region-specific upregulation of EphA5 mRNA and protein expression in the developing mouse brain. The GnRH neurons in GNR23 mice overexpressed EphA5 from embryonic day 11, whereas ephrin A3 and A5 mRNA levels in olfactory neurons were unchanged. The GnRH neurons were found to be slow in commencing their migration from the olfactory placode and also to form abnormal clusters of cells on the olfactory axons, prohibiting their migration out of the nose. As a result, adult hemizygous mice had only 40% of the normal complement of GnRH neurons in the brain, whereas homozygous mice had <15%. This resulted in infertility in adult female homozygous GNR23 mice, suggesting that some cases of human hypogonadotropic hypogonadism may result from ephrin-related mutations. These data provide evidence for a role of EphA-ephrin signaling in the axophilic migration of the GnRH neurons during embryogenesis.

Tissue and plasma expression of the angiogenic peptide adrenomedullin in breast cancer.

Adrenomedullin (ADM) is an angiogenic factor that has also been shown to be a mitogen and a hypoxia survival factor for tumour cells. These properties point to ADM as a potential promoter of human malignancies, but little data are available concerning the expression of ADM in human breast cancer. In the present work, we have examined ADM peptide expression in a series of malignant breast tumours by immunohistochemistry using a newly developed anti-ADM monoclonal antibody. In addition, ADM plasma concentrations in breast cancer patients and healthy controls were determined by radioimmunoassay. Of the examined breast cancer samples, 27/33 (82%) showed a moderate to strong staining intensity. ADM-peptide expression in breast tumours was significantly correlated with axillary lymph node metastasis (P=0.030). Analysis of ADM plasma concentrations showed no significant difference between the circulating ADM levels of breast cancer patients and healthy controls. However, a significant positive correlation was found between tumour size and plasma ADM levels (r=0.641, P=0.017). Moreover, ADM levels in breast cancer patients correlated with the presence of lymph node metastasis (P=0.002). In conclusion, we have shown for the first time that ADM peptide is widely expressed in breast cancer and that the degree of expression is associated with lymph node metastasis. ADM peptide in plasma of breast cancer patients reflects the size of the primary tumour, but is unlikely to be a useful tumour marker for the detection of breast cancer. Plasma ADM might represent an independent predictor of lymph node metastasis. The clinical implications of these findings remain to be evaluated.