Tumour extracellular vesicles and particles induce liver metabolic dysfunction.

Cancer alters the function of multiple organs beyond those targeted by metastasis1,2. Here we show that inflammation, fatty liver and dysregulated metabolism are hallmarks of systemically affected livers in mouse models and in patients with extrahepatic metastasis. We identified tumour-derived extracellular vesicles and particles (EVPs) as crucial mediators of cancer-induced hepatic reprogramming, which could be reversed by reducing tumour EVP secretion via depletion of Rab27a. All EVP subpopulations, exosomes and principally exomeres, could dysregulate hepatic function. The fatty acid cargo of tumour EVPs-particularly palmitic acid-induced secretion of tumour necrosis factor (TNF) by Kupffer cells, generating a pro-inflammatory microenvironment, suppressing fatty acid metabolism and oxidative phosphorylation, and promoting fatty liver formation. Notably, Kupffer cell ablation or TNF blockade markedly decreased tumour-induced fatty liver generation. Tumour implantation or pre-treatment with tumour EVPs diminished cytochrome P450 gene expression and attenuated drug metabolism in a TNF-dependent manner. We also observed fatty liver and decreased cytochrome P450 expression at diagnosis in tumour-free livers of patients with pancreatic cancer who later developed extrahepatic metastasis, highlighting the clinical relevance of our findings. Notably, tumour EVP education enhanced side effects of chemotherapy, including bone marrow suppression and cardiotoxicity, suggesting that metabolic reprogramming of the liver by tumour-derived EVPs may limit chemotherapy tolerance in patients with cancer. Our results reveal how tumour-derived EVPs dysregulate hepatic function and their targetable potential, alongside TNF inhibition, for preventing fatty liver formation and enhancing the efficacy of chemotherapy.

Tumor-derived nanoseeds condition the soil for metastatic organotropism.

Primary tumors secrete a variety of factors to turn distant microenvironments into favorable and fertile 'soil' for subsequent metastases. Among these 'seeding' factors that initiate pre-metastatic niche (PMN) formation, tumor-derived extracellular vesicles (EVs) are of particular interest as tumor EVs can direct organotropism depending on their surface integrin profiles. In addition, EVs also contain versatile, bioactive cargo, which include proteins, metabolites, lipids, RNA, and DNA fragments. The cargo incorporated into EVs is collectively shed from cancer cells and cancer-associated stromal cells. Increased understanding of how tumor EVs promote PMN establishment and detection of EVs in bodily fluids highlight how tumor EVs could serve as potential diagnostic and prognostic biomarkers, as well as provide a therapeutic target for metastasis prevention. This review focuses on tumor-derived EVs and how they direct organotropism and subsequently modulate stromal and immune microenvironments at distal sites to facilitate PMN formation. We also outline the progress made thus far towards clinical applications of tumor EVs.

Treatment of chronic symptoms following mild traumatic brain injury with transcranial LED: a sham run-in pilot study of photobiomodulation therapy.

PRIMARY OBJECTIVE: We evaluated whether photobiomodulation with red/near infrared light applied transcranially via light emitting diodes (LED) was associated with reduced symptoms and improved cognitive functioning in patients with chronic symptoms following mild traumatic brain injury. RESEARCH DESIGN: Participants (3 men, 6 women; 22-61 years-old) underwent a 6-week intervention involving 18 40-minute transcranial LED treatment sessions. METHODS AND PROCEDURES: Reliable change indices were calculated for 10 neuropsychological test scores and 3 self-report questionnaires of subjective cognition, post-concussion symptoms, and depression at baseline and following treatment. Questionnaires were also administered after 2-week sham and at 1-month and 2-month follow-ups. MAIN OUTCOME AND RESULTS: Only 2 participants improved on neuropsychological testing. On questionnaires, 4 reported improved cognition, 5 reported improved post-concussion symptoms, and 3 reported improved depression. Significant improvement in 2 or more domains was reported by 4 participants and mostly maintained at both follow-ups. CONCLUSIONS: Most participants did not improve on neuropsychological testing. A minority self-reported improvement in symptoms, potentially explained by the intervention, psychiatric medication changes, placebo effects, or other factors. Selecting participants with different clinical characteristics, and dosing and delivery system changes, may produce different results. A study design accounting for placebo effects appears warranted in future trials.

Huperzine A for the treatment of cognitive, mood, and functional deficits after moderate and severe TBI (HUP-TBI): results of a Phase II randomized controlled pilot study: implications for understanding the placebo effect.

Objective: To investigate the effect of Huperzine A on memory and learning in individuals with moderate-severe traumatic brain injury (TBI).Design: Randomized, double-blind, placebo-controlled Phase II clinical trial.Methods: Subjects were randomly assigned to receive Huperzine A or placebo for 12 weeks and were assessed during in-person visits at screening/baseline, and 6, 12, 24, and 52 weeks post-injury. Changes in memory and learning scores on the California Verbal Learning Test - 2nd Edition (CVLT-II) from baseline to week 12 were assessed using permutation tests and regression analyses.Results: There was no difference between the Huperzine A and placebo groups in memory performance after 12 weeks of treatment. In the placebo group, significant improvements were noted in learning and memory scores. Both groups showed clinically important improvements in depression on the Beck Depression Index.Conclusions: The clinically important improvements in cognitive and emotional outcomes observed in both the placebo and active treatment arms of this clinical trial of Huperzine A are best understood in the context of a placebo effect. Future trials involving patients with moderate-severe TBI in the subacute to chronic phases of recovery should be designed to account for placebo effects as failure to do so may lead to spurious conclusions.

Biopolymer Molecular Weight Can Modulate the Wound Healing Efficacy of Multivalent Sonic Hedgehog-Hyaluronic Acid Conjugates.

There is a clinical need for new therapeutics to improve healing of chronic impaired wounds. Thus, we investigated how biopolymer conjugation could be used to improve the wound healing performance of a key growth factor for tissue regeneration: Sonic hedgehog (Shh). We generated two multivalent Shh conjugates (mvShh) using hyaluronic acid with two different MWs, which exhibited equivalent potency and proteolytic protection in vitro. Using db/db diabetic mice, we showed that mvShh made with smaller HyA MW resulted in more rapid and robust neovascularization compared to mvShh made with larger MW HyA. Further, smaller mvShh conjugates resulted in faster wound resolution compared to the unconjugated Shh. This study is the first to show how the wound healing efficacy of multivalent protein-polymer conjugates is sensitive to the polymer MW, and our findings suggest that this parameter could be used to enhance the efficacy of growth factor conjugates.

Tumour-derived Extracellular Vesicle and Particle Reprogramming of Interstitial Macrophages in the Lung Pre-Metastatic Niche Enhances Vascular Permeability and Metastatic Potential.

Extracellular vesicles and particles (EVPs) are pivotal mediators of pre-metastatic niche formation and cancer progression, including induction of vascular permeability, which facilitates tumor cell extravasation and metastasis. However, the mechanisms through which EVPs exert this effect remain poorly understood. Here, we elucidate a novel mechanism by which tumor EVPs enhance endothelial cell permeability, tumor extravasation, and lung metastasis to different degrees, depending on tumor type. Strikingly, vascular leakiness is observed within 48h following tumor implantation and as early as one hour following intravenous injection of tumour-derived EVPs in naïve mice. Surprisingly, rather than acting directly on endothelial cells, EVPs first activate interstitial macrophages (IMs) leading to activation of JAK/STAT signaling and IL-6 secretion in IMs which subsequently promote endothelial permeability. Depletion of IMs significantly reduces tumour-derived EVP-dependent vascular leakiness and metastatic potential. Tumour EVPs that strongly induce vascular leakiness express high levels of ITGα5, and ITGα5 ablation impairs IM activation, cytokine secretion, and subsequently vascular permeability and metastasis. Importantly, IL-6 expression is elevated in IMs from non-involved tumor-adjacent lung tissue compared to distal lung tissue in lung cancer patients, highlight the clinical relevance of our discovery. Our findings identify a key role for IM activation as an initiating step in tumor type-specific EVP-driven vascular permeability and metastasis, offering promising targets for therapeutic intervention.

Multi-parametric atlas of the pre-metastatic liver for prediction of metastatic outcome in early-stage pancreatic cancer.

Metastasis occurs frequently after resection of pancreatic cancer (PaC). In this study, we hypothesized that multi-parametric analysis of pre-metastatic liver biopsies would classify patients according to their metastatic risk, timing and organ site. Liver biopsies obtained during pancreatectomy from 49 patients with localized PaC and 19 control patients with non-cancerous pancreatic lesions were analyzed, combining metabolomic, tissue and single-cell transcriptomics and multiplex imaging approaches. Patients were followed prospectively (median 3 years) and classified into four recurrence groups; early (<6 months after resection) or late (>6 months after resection) liver metastasis (LiM); extrahepatic metastasis (EHM); and disease-free survivors (no evidence of disease (NED)). Overall, PaC livers exhibited signs of augmented inflammation compared to controls. Enrichment of neutrophil extracellular traps (NETs), Ki-67 upregulation and decreased liver creatine significantly distinguished those with future metastasis from NED. Patients with future LiM were characterized by scant T cell lobular infiltration, less steatosis and higher levels of citrullinated H3 compared to patients who developed EHM, who had overexpression of interferon target genes (MX1 and NR1D1) and an increase of CD11B+ natural killer (NK) cells. Upregulation of sortilin-1 and prominent NETs, together with the lack of T cells and a reduction in CD11B+ NK cells, differentiated patients with early-onset LiM from those with late-onset LiM. Liver profiles of NED closely resembled those of controls. Using the above parameters, a machine-learning-based model was developed that successfully predicted the metastatic outcome at the time of surgery with 78% accuracy. Therefore, multi-parametric profiling of liver biopsies at the time of PaC diagnosis may determine metastatic risk and organotropism and guide clinical stratification for optimal treatment selection.

Advancement in Cellular Topographic and Nanoparticle Capture Imaging by High Resolution Microscopy Incorporating a Freeze-Drying and Gaseous Nitrogen-based Approach.

Scanning electron microscopy (SEM) offers an unparalleled view of the membrane topography of mammalian cells by using a conventional osmium (OsO4) and ethanol-based tissue preparation. However, conventional SEM methods limit optimal resolution due to ethanol and lipid interactions and interfere with visualization of fluorescent reporter proteins. Therefore, SEM correlative light and electron microscopy (CLEM) has been hindered by the adverse effects of ethanol and OsO4 on retention of fluorescence signals. To overcome this technological gap in achieving high-resolution SEM and retain fluorescent reporter signals, we developed a freeze-drying method with gaseous nitrogen (FDGN). We demonstrate that FDGN preserves cyto-architecture to allow visualization of detailed membrane topography while retaining fluorescent signals and that FDGN processing can be used in conjunction with a variety of high-resolution imaging systems to enable collection and validation of unique, high-quality data from these approaches. In particular, we show that FDGN coupled with high resolution microscopy provided detailed insight into viral or tumor-derived extracellular vesicle (TEV)-host cell interactions and may aid in designing new approaches to intervene during viral infection or to harness TEVs as therapeutic agents.

Development and acceptability of a mobile health application integrated with the electronic heath record for treatment of chronic insomnia disorder.

STUDY OBJECTIVES: To describe the development and feasibility of a cognitive behavioral therapy for insomnia (CBT-I) program delivered via personal digital devices and fully integrated with the electronic health record (EHR). METHODS: A multidisciplinary team of clinicians and members of our Center for Digital Health collaborated to develop a Chronic Insomnia Interactive Care Plan (ChI-ICP), an application that provides personalized and just in time education and promotes self-management using CBT-I concepts, and is activated from and fully integrated into the EHR. Following development, we evaluated patient engagement and workflows, assessed changes to provider workload, and examined outcomes on measures of insomnia during a pilot deployment of the application. RESULTS: A total of 222 patients were enrolled and 179 engaged with the plan during the 3-month pilot program. Enrolled patients generated an average of 3.9 ± 2.3 In Basket messages, most being automated notifications related to noncompletion of assigned tasks, while only a few were related to patients requesting additional training or help with insomnia. Sleep efficiency improved from baseline until the completion of the program from 74.5% ± 16.7% to 87.6% ± 10.8% (P = .001), and the Insomnia Severity Index improved from 14.9 ± 5.22 to 11.6 ± 4.80 (P = .006). CONCLUSIONS: In this pilot implementation of an integrated ChI-ICP, patient engagement was favorable, workflows and workload were not significantly burdensome for the care teams, and initial evaluation of efficacy was favorable. This provides evidence for an application that is a scalable method to assist patients with chronic insomnia and future work should assess its efficacy in controlled trials. CITATION: Morgenthaler TI, Kolla BP, Anderson SE, et al. Development and acceptability of a mobile health application integrated with the electronic heath record for treatment of chronic insomnia disorder. J Clin Sleep Med. 2022;18(12):2785-2792.

HOXA3 modulates injury-induced mobilization and recruitment of bone marrow-derived cells.

The regulated recruitment and differentiation of multipotent bone marrow-derived cells (BMDCs) to sites of injury are critical for efficient wound healing. Previously we demonstrated that sustained expression of HOXA3 both accelerated wound healing and promoted angiogenesis in diabetic mice. In this study, we have used green fluorescent protein-positive bone marrow chimeras to investigate the effect of HOXA3 expression on recruitment of BMDCs to wounds. We hypothesized that the enhanced neovascularization induced by HOXA3 is due to enhanced mobilization, recruitment, and/or differentiation of BMDCs. Here we show that diabetic mice treated with HOXA3 displayed a significant increase in both mobilization and recruitment of endothelial progenitor cells compared with control mice. Importantly, we also found that HOXA3-treated mice had significantly fewer inflammatory cells recruited to the wound compared with control mice. Microarray analyses of HOXA3-treated wounds revealed that indeed HOXA3 locally increased expression of genes that selectively promote stem/progenitor cell mobilization and recruitment while also suppressing expression of numerous members of the proinflammatory nuclear factor kappaB pathway, including myeloid differentiation primary response gene 88 and toll-interacting protein. Thus HOXA3 accelerates wound repair by mobilizing endothelial progenitor cells and attenuating the excessive inflammatory response of chronic wounds.

Restoring transcription factor HoxA5 expression inhibits the growth of experimental hemangiomas in the brain.

Hemangiomas are angiogenesis-dependent benign vascular tumors that can rupture and cause intracranial hemorrhages. We previously showed that the transcription factor homeobox A5 (HoxA5), which is absent in activated angiogenic endothelial cells can block angiogenesis. Here, we investigated whether restoring expression of HoxA5 blocks hemangioma growth by transplanting mouse hemangioendothelioma endothelial cells (EOMA) or HoxA5-expressing EOMA cells into the brains of mice. The EOMA cells induced brain hemangiomas characterized by large cystlike spaces lined by thin walls of endothelial cells surrounded by scant smooth muscle cells. When HoxA5-expressing EOMA cells were injected, lesion volumes were reduced between 5- and 20-fold compared with the EOMA control group (p < 0.05). Restoration of HoxA5 was associated with increased thrombospondin-2, which inhibits angiogenesis and reduced hypoxia-inducible factor 1alpha expression. These data suggest that restoring HoxA5 can attenuate experimental brain hemangioma development.

Neovascularization of ischemic tissues by gene delivery of the extracellular matrix protein Del-1.

The ECM protein Del-1 is one of several novel ECM proteins that accumulate around angiogenic blood vessels in embryonic and tumor tissue and promote angiogenesis in the absence of exogenous growth factors. Del-1 expressed in mouse or rabbit ischemic hind-limb muscle by gene transfer rapidly promotes new blood vessel formation and restores muscle function. This angiogenic ECM protein initiates angiogenesis by binding to integrin alphavbeta5 on resting endothelium, thereby resulting in expression of the transcription factor Hox D3 and integrin alphavbeta3. Hox D3 converts resting endothelium to angiogenic endothelium by inducing expression of proangiogenic molecules such as integrin alphavbeta3. These findings provide evidence for an angiogenic switch that can be initiated in the absence of exogenous growth factors and indicate that the angiogenic matrix protein Del-1 may be a useful tool for the therapy of ischemic disease.

Homeobox D10 induces phenotypic reversion of breast tumor cells in a three-dimensional culture model.

Homeobox (Hox) genes are master regulatory genes that direct organogenesis and maintain differentiated tissue function. We previously reported that HoxD10 helps to maintain a quiescent, differentiated phenotype in endothelial cells by suppressing expression of genes involved in remodeling the extracellular matrix and cell migration. Here we investigated whether HoxD10 could also promote or maintain a differentiated phenotype in epithelial cells. We observed that HoxD10 expression is progressively reduced in epithelial cells as malignancy increases in both breast and endometrial tumors. Retroviral gene transfer to restore expression of HoxD10 in the malignant breast tumor cells MDA-MB-231 significantly impaired migration, and when these cells were cultured in a three-dimensional laminin-rich basement membrane (3DlrBM) model, they formed polarized, acinar structures. This phenotypic reversion was accompanied by decreased alpha3 integrin expression and reduced proliferation. Importantly, expression of HoxD10 in the MDA-MB-231 cells inhibited their ability to form tumors in mouse xenografts. Taken together, our results suggest that HoxD10 has tumor-suppressive functions for mammary epithelial cells.

The tumor promoter-activated protein kinase Cs are a system for regulating filopodia.

Different protein kinase C (PKC) isoforms have distinct roles in regulating cell functions. The conventional (α, ß, γ) and novel (δ, ɛ, η, θ) classes are targets of phorbol ester tumor promoters, which are surrogates of endogenous second messenger, diacylglycerol. The promoter-stimulated disappearance of filopodia was investigated by use of blocking peptides (BPs) that inhibit PKC maturation and/or docking. Filopodia were partially rescued by a peptide representing PKC ɛ hydrophobic sequence, but also by a myristoylated PKC α/ß pseudosubstrate sequence, and an inhibitor of T-cell protein tyrosine phosphatase (TC-PTP). The ability to turn over filopodia was widely distributed among PKC isoforms. PKC α and η hydrophobic sequences enhanced filopodia in cells in the absence of tumor promoter treatment. With transcriptional knockdown of PKC α, the content of PKC ɛ predominated over other isoforms. PKC ɛ could decrease filopodia significantly in promoter-treated cells, and this was attributed to ruffling. The presence of PKC α counteracted the PKC ɛ-mediated enhancement of ruffling. The results showed that there were two mechanisms of filopodia downregulation. One operated in the steady-state and relied on PKC α and η. The other was stimulated by tumor promoters and relied on PKC ɛ. Cycles of protrusion and retraction are characteristic of filopodia and are essential for the cell to orient itself during chemotaxis and haptotaxis. By suppressing filopodia, PKC ɛ can create a long-term "memory" of an environmental signal that may act in nature as a mnemonic device to mark the direction of a repulsive signal.

Alk5 inhibition increases delivery of macromolecular and protein-bound contrast agents to tumors.

Limited transendothelial permeability across tumor microvessels represents a significant bottleneck in the development of tumor-specific diagnostic agents and theranostic drugs. Here, we show an approach to increase transendothelial permeability of macromolecular and nanoparticle-based contrast agents via inhibition of the type I TGF-ß receptor, activin-like kinase 5 (Alk5), in tumors. Alk5 inhibition significantly increased tumor contrast agent delivery and enhancement on imaging studies, while healthy organs remained relatively unaffected. Imaging data correlated with significantly decreased tumor interstitial fluid pressure, while tumor vascular density remained unchanged. This immediately clinically translatable concept involving Alk5 inhibitor pretreatment prior to an imaging study could be leveraged for improved tumor delivery of macromolecular and nanoparticle-based imaging probes and, thereby, facilitate development of more sensitive imaging tests for cancer diagnosis, enhanced tumor characterization, and personalized, image-guided therapies.

Sulf-2, a proangiogenic heparan sulfate endosulfatase, is upregulated in breast cancer.

Sulf-2 is an endosulfatase with activity against glucosamine-6-sulfate modifications within subregions of intact heparin. The enzyme has the potential to modify the sulfation status of extracellular heparan sulfate proteoglycan (HSPG) glycosaminoglycan chains and thereby to regulate interactions with HSPG-binding proteins. In the present investigation, data mining from published studies was employed to establish Sulf-2 mRNA upregulation in human breast cancer. We further found that cultured breast carcinoma cells expressed Sulf-2 mRNA and released enzymatically active proteins into conditioned medium. In two mouse models of mammary carcinoma, Sulf-2 mRNA was upregulated in comparison to its expression in normal mammary gland. Although mRNA was present in normal tissues, Sulf-2 protein was undetectable; it was, however, detected in some premalignant lesions and in tumors. The protein was localized to the epithelial cells of the tumors. In support of the possible mechanistic relevance of Sulf-2 upregulation in tumors, purified recombinant Sulf-2 promoted angiogenesis in the chick chorioallantoic membrane assay.

Organized living: from cell surfaces to basement membranes.

Binding of extracellular matrix (ECM) proteins to integrin receptors initiates intracellular signaling events that are essential for the differentiation and survival of epithelial cells. However, the propagation and processing of these signals also depend on the cells acquiring an appropriate three-dimensional morphology and polarity after contact with the ECM. In fact, even if adhesion to the ECM is maintained but subsequent cellular organization and polarity are impaired, epithelial cells fail to fully differentiate and become susceptible to apoptotic stimuli. Studies using three-dimensional tissue culture models with reconstituted basement membranes not only demonstrate the central role of tissue organization for differentiation and survival, but also emphasize how acquiring this organized polarized phenotype can override a number of genetic changes that would otherwise disrupt normal tissue function.

A role for Hox A5 in regulating angiogenesis and vascular patterning.

BACKGROUND: Homeobox (Hox) genes are transcriptional regulators which modulate embryonic morphogenesis and pathological tissue remodeling in adults via regulation of genes associated with cell-cell or cell extracellular matrix (ECM) interactions. We previously showed that while Hox 3 genes promote angiogenesis, Hox D10 inhibits this process. METHODS AND RESULTS: Here we show that another Hox family gene, Hox A5, also blocks angiogenesis but accomplishes this by targeting different downstream genes than Hox D10. Sustained expression of Hox A5 leads to down regulation of many pro-angiogenic genes including VEGFR2, ephrin A1, Hif1alpha and COX-2. In addition, Hox A5 also upregulates expression of anti-angiogenic genes including Thrombospondin-2. Furthermore, we show that while Hox A5 mRNA is expressed in quiescent endothelial cells (EC), its expression is diminished or absent in active angiogenic EC found in association with breast tumors or in proliferating infantile hemangiomas. CONCLUSIONS: Together our results suggest that restoring Hox A5 expression may provide a novel means to limit breast tumor growth or expansion of hemangiomas.

Comparing Rasch analyses probability estimates to sensitivity, specificity and likelihood ratios when examining the utility of medical diagnostic tests.

INTRODUCTION: Medical diagnostic tests are evaluated based on measures of sensitivity (Sn), specificity (Sp), and likelihood ratios (LR). These procedures are limited in the event of a biased gold standard or missing data. Interpretations of these measures are frequently inappropriate. PURPOSE: The Rasch measurement model (RMM) was examined as a method to provide evidence of diagnostic test utility in order to overcome the limitations of Sn, Sp, and LR. METHODS: Patients suspected of a knee ligament tear (n = 825) were studied, by evaluating four diagnostic tests. The RMM probability estimates for each test were compared to estimates of Sn, Sp, and LR. RESULTS: The RMM provided probability estimates for the diagnosis that were comparable to likelihood ratios. These probability estimates correlated with the estimates of Sn, Sp, and LR. The RMM estimates were not affected by missing data. DISCUSSION: The RMM may provide an alternative means to study the utility of medical diagnostic tests to estimate the probability of disease presence/absence.

Sustained endothelial expression of HoxA5 in vivo impairs pathological angiogenesis and tumor progression.

HoxA5 is expressed in quiescent endothelial cells (EC), but absent in activated angiogenic EC. To examine the efficacy of targeting HoxA5 therapeutically to quell pathologic or tumor angiogenesis, we generated an inducible, transgenic mouse model of sustained HoxA5 expression in ECs. During pathologic angiogenesis, sustained HoxA5 regulates expression several angiogenic effector molecules, notably increased expression of TSP-2 and reduced expression of VEGF, thus leading to inhibition of pathological angiogenesis in tissues. To evaluate if this impressive reduction of vascularization could also impact tumor angiogenesis, HoxA5 mice were bred with a mouse model of de novo squamous carcinogenesis, e.g., K14-HPV16 mice. Activation of EC-HoxA5 significantly reduced infiltration by mast cells into neoplastic skin, an early hallmark of progression to dysplasia, reduced angiogenic vasculature, and blunted characteristics of tumor progression. To evaluate HoxA5 as a therapeutic, topical application of a HoxA5 transgene onto early neoplastic skin of K14-HPV16 mice similarly resulted in a significant impairment of angiogenic vasculature and progression to dysplasia to a similar extent as observed with genetic delivery of HoxA5. Together these data indicate that HoxA5 represents a novel molecule for restricting pathological and tumorigenic angiogenesis.