Angiogenesis is an essential component of normal wound repair, yet the primary mediators of wound angiogenesis have not been well described. The current study characterizes the contribution of vascular endothelial cell growth factor (VEGF) to the angiogenic environment of human surgical wounds. Surgical wound fluid samples (n = 70) were collected daily for up to 7 postoperative days (POD) from 14 patients undergoing mastectomy or neck dissection. VEGF levels in surgical wound fluid were lowest on POD 0, approximating values of serum, but increased steadily through POD 7. An opposite pattern was noted for basic fibroblast growth factor-2. Fibroblast growth factor-2, which has been previously described as a wound angiogenic factor, exhibited highest levels at POD 0, declining to near serum levels by POD 3. Surgical wound fluid form all time points stimulated marked endothelial cell chemotaxis and induced a brisk neovascular response in the rat corneal micropocket angiogenesis assay. Antibody neutralization of VEGF did not affect the in vitro chemotactic or the in vivo angiogenic activity early wound samples (POD 0). In contrast, VEGF neutralization significantly attenuated both chemotactic activity (mean decrease 76 +/- 13%, P < 0.01) and angiogenic activity (5 of 5 samples affected) of later wound samples (POD 3 and 6). The results suggest a model of wound angiogenesis in which an initial angiogenic stimulus is supplied by fibroblast growth factor-2, followed by a subsequent and more prolonged angiogenic stimulus mediated by VEGF.

Thalidomide (Thal) can overcome drug resistance in multiple myeloma (MM) but is associated with somnolence, constipation, and neuropathy. In previous in vitro studies, we have shown that the potent immunomodulatory derivative of thalidomide (IMiD) CC-5013 induces apoptosis or growth arrest even in resistant MM cell lines and patient cells, decreases binding of MM cells to bone marrow stromal cells (BMSCs), inhibits the production in the BM milieu of cytokines (interleukin-6 [IL-6], vascular endothelial growth factor [VEGF], tumor necrosis factor-alpha [TNF-alpha]) mediating growth and survival of MM cells, blocks angiogenesis, and stimulates host anti-MM natural killer (NK) cell immunity. Moreover, CC-5013 also inhibits tumor growth, decreases angiogenesis, and prolongs host survival in a human plasmacytoma mouse model. In the present study, we carried out a phase 1 CC-5013 dose-escalation (5 mg/d, 10 mg/d, 25 mg/d, and 50 mg/d) study in 27 patients (median age 57 years; range, 40-71 years) with relapsed and refractory relapsed MM. They received a median of 3 prior regimens (range, 2-6 regimens), including autologous stem cell transplantation and Thal in 15 and 16 patients, respectively. In 24 evaluable patients, no dose-limiting toxicity (DLT) was observed in patients treated at any dose level within the first 28 days; however, grade 3 myelosuppression developed after day 28 in all 13 patients treated with 50 mg/d CC-5013. In 12 patients, dose reduction to 25 mg/d was well tolerated and therefore considered the maximal tolerated dose (MTD). Importantly, no significant somnolence, constipation, or neuropathy has been seen in any cohort. Best responses of at least 25% reduction in paraprotein occurred in 17 (71%) of 24 patients (90% confidence interval [CI], 52%-85%), including 11 (46%) patients who had received prior Thal. Stable disease (less than 25% reduction in paraprotein) was observed in an additional 2 (8%) patients. Therefore, 17 (71%) of 24 patients (90% CI, 52%-85%) demonstrated benefit from treatment. Our study therefore provides the basis for the evaluation of CC-5013, either alone or in combination, to treat patients with MM at earlier stages of disease.

The vertebrate body contains an extensive blood vessel network that forms, with a few exceptions, by endothelial sprouting from the existing vasculature. This process, termed angiogenesis, involves complex and highly dynamic interactions between endothelial cells and their environment. Pro-angiogenic signals, such as VEGF, promote endothelial motility, filopodia extension and proliferation, and, together with Notch signaling, controls whether specific endothelial cells become lead tip cells or trailing stalk cells. Sprouts then convert into endothelial tubules and form connections with other vessels, which requires the local suppression of motility and the formation of new cell-cell junctions. We here review the dynamics of angiogenesis in the context of key molecules and pathways controlling tip cell selection, sprouting and the formation of new vessels.

In inflammatory CNS conditions such as multiple sclerosis (MS), current options to treat clinical relapse are limited, and more selective agents are needed. Disruption of the blood-brain barrier (BBB) is an early feature of lesion formation that correlates with clinical exacerbation, leading to edema, excitotoxicity, and entry of serum proteins and inflammatory cells. Here, we identify astrocytic expression of VEGF-A as a key driver of BBB permeability in mice. Inactivation of astrocytic Vegfa expression reduced BBB breakdown, decreased lymphocyte infiltration and neuropathology in inflammatory and demyelinating lesions, and reduced paralysis in a mouse model of MS. Knockdown studies in CNS endothelium indicated activation of the downstream effector eNOS as the principal mechanism underlying the effects of VEGF-A on the BBB. Systemic administration of the selective eNOS inhibitor cavtratin in mice abrogated VEGF-A-induced BBB disruption and pathology and protected against neurologic deficit in the MS model system. Collectively, these data identify blockade of VEGF-A signaling as a protective strategy to treat inflammatory CNS disease.

The angiogenic sprout has been compared to the growing axon, and indeed, many proteins direct pathfinding by both structures. The Roundabout (Robo) proteins are guidance receptors with well-established functions in the nervous system; however, their role in the mammalian vasculature remains ill defined. Here we show that an endothelial-specific Robo, Robo4, maintains vascular integrity. Activation of Robo4 by Slit2 inhibits vascular endothelial growth factor (VEGF)-165-induced migration, tube formation and permeability in vitro and VEGF-165-stimulated vascular leak in vivo by blocking Src family kinase activation. In mouse models of retinal and choroidal vascular disease, Slit2 inhibited angiogenesis and vascular leak, whereas deletion of Robo4 enhanced these pathologic processes. Our results define a previously unknown function for Robo receptors in stabilizing the vasculature and suggest that activating Robo4 may have broad therapeutic application in diseases characterized by excessive angiogenesis and/or vascular leak.

BACKGROUND

Cell therapy with bone marrow-derived stem/progenitor cells is a novel option for improving neovascularization and cardiac function in ischemic heart disease. Circulating endothelial progenitor cells in patients with coronary heart disease are impaired with respect to number and functional activity. However, whether this impairment also extends to bone marrow-derived mononuclear cells (BM-MNCs) in patients with chronic ischemic cardiomyopathy (ICMP) is unclear.

RESULTS

BM-MNCs were isolated from bone marrow aspirates in 18 patients with ICMP (ejection fraction, 38+/-11%) and 8 healthy control subjects (controls). The number of hematopoietic stem/progenitor cells (CD34+/CD133+), CD49d(+) (VLA-4) cells, and CXCR4+ cells did not differ between the 2 groups. However, the colony-forming capacity of BM-MNCs from patients with ICMP was significantly lower compared with BM-MNCs from healthy controls (37.3+/-25.0 versus 113.8+/-70.4 granulocyte-macrophage colony-forming units; P=0.009). Likewise, the migratory response to stromal cell-derived factor 1 (SDF-1) and vascular endothelial growth factor (VEGF) was significantly reduced in BM-MNCs derived from patients with ICMP compared with BM-MNCs from healthy controls (SDF-1, 46.3+/-26.2 versus 108.6+/-40.4 cells/microscopic field, P<0.001; VEGF, 34+/-24.2 versus 54.8+/-29.3 cells/microscopic field, P=0.027). To assess the in vivo relevance of these findings, we tested the functional activity of BM-MNCs to improve neovascularization in a hindlimb animal model using nude mice. Two weeks after ligation of the femoral artery and intravenous injection of 5x10(5) BM-MNCs, laser Doppler-derived relative limb blood flow in mice treated with BM-MNCs from patients with ICMP was significantly lower compared with mice treated with BM-MNCs from healthy controls (0.45+/-0.14 versus 0.68+/-0.15; P<0.001). The in vivo neovascularization capacity of BM-MNCs closely correlated with the in vitro assessment of SDF-1-induced migration (r=0.78; P<0.001) and colony-forming capacity (r=0.74; P<0.001).

CONCLUSIONS

BM-MNCs isolated from patients with ICMP have a significantly reduced migratory and colony-forming activity in vitro and a reduced neovascularization capacity in vivo despite similar content of hematopoietic stem cells. This functional impairment of BM-MNCs from patients with ICMP may limit their therapeutic potential for clinical cell therapy.

Vascular endothelial growth factors (VEGFs) belong to the platelet-derived growth factor supergene family, and they play central roles in the regulation of angiogenesis and lymphangiogenesis. VEGF-A, the major factor for angiogenesis, binds to two tyrosine kinase (TK) receptors, VEGFR-1 (Flt-1) and VEGFR-2 (KDR/Flk-1), and regulates endothelial cell proliferation, migration, vascular permeability, secretion and other endothelial functions. VEGFR-2 exhibits a strong TK activity towards pro-angiogenic signals, whereas the soluble VEGFR-1 (sFlt-1) functions as an endogenous VEGF inhibitor. sFlt-1 is abnormally overexpressed in the placenta of preeclampsia patients, resulting in the major symptoms of the disease due to abnormal trapping of VEGFs. The VEGF-VEGFR system is crucial for tumour angiogenesis, and anti-VEGF-VEGFR molecules are now widely used in the clinical field to treat cancer patients. The efficacy of these molecules in prolonging the overall survival of patients has been established; however, some cancers do not respond well and reduced tumour sensitivity to anti-VEGF signals may occur after long-term treatment. The molecular basis of tumour refractoriness should be determined to improve anti-angiogenic therapy.

Targeted delivery of RNA-based therapeutics for cancer therapy remains a challenge. We have developed a LPH (liposome-polycation-hyaluronic acid) nanoparticle formulation modified with tumor-targeting single-chain antibody fragment (scFv) for systemic delivery of small interfering RNA (siRNA) and microRNA (miRNA) into experimental lung metastasis of murine B16F10 melanoma. The siRNAs delivered by the scFv targeted nanoparticles efficiently downregulated the target genes (c-Myc/MDM2/VEGF) in the lung metastasis. Two daily intravenous injections of the combined siRNAs in the GC4-targeted nanoparticles significantly reduced the tumor load in the lung. miRNA-34a (miR-34a) induced apoptosis, inhibited survivin expression, and downregulated MAPK pathway in B16F10 cells. miR-34a delivered by the GC4-targeted nanoparticles significantly downregulated the survivin expression in the metastatic tumor and reduced tumor load in the lung. When miR-34a and siRNAs were co-formulated in GC4-targeted nanoparticles, an enhanced anticancer effect was observed.

Four decades ago, angiogenesis was recognized as a therapeutic target for blocking cancer growth. Because of its importance, VEGF has been at the center stage of antiangiogenic therapy. Now, several years after FDA approval of an anti-VEGF antibody as the first antiangiogenic agent, many patients with cancer and ocular neovascularization have benefited from VEGF-targeted therapy; however, this anticancer strategy is challenged by insufficient efficacy, intrinsic refractoriness, and resistance. Here, we examine recent discoveries of new mechanisms underlying angiogenesis, discuss successes and challenges of current antiangiogenic therapy, and highlight emerging antiangiogenic paradigms.

The onset of hematopoiesis in the mouse embryo and in the embryonic stem (ES) cell differentiation model is defined by the emergence of the hemangioblast, a progenitor with both hematopoietic and vascular potential. While there is evidence for the existence of a hemangioblast in the mouse, it is unclear if this progenitor develops during the establishment of the human hematopoietic system. In this report, we have mapped hematopoietic development in human ES cell (hESC) differentiation cultures and demonstrated that a comparable hemangioblast population exists. The human hemangioblasts were identified by their capacity to generate blast colonies that display both hematopoietic and vascular potential. These colony-forming cells express the receptor tyrosine kinase KDR (VEGF receptor 2) and represent a transient population that develops in BMP-4-stimulated embryoid bodies (EBs) between 72 and 96 hours of differentiation, prior to the onset of the primitive erythroid program. Two distinct types of hemangioblasts were identified, those that give rise to primitive erythroid cells, macrophages, and endothelial cells and those that generate only the primitive erythroid population and endothelial cells. These findings demonstrate for the first time the existence of the human hemangioblast and in doing so identify the earliest stage of hematopoietic commitment.

Vascular endothelial growth factor (VEGF) may have a physiologic role in regulating vessel permeability and contributes to the pathophysiology of diabetic retinopathy as well as tumor development. We set out to ascertain the mechanism by which VEGF regulates paracellular permeability in rats. Intra-ocular injection of VEGF caused a post-translational modification of occludin as determined by a gel shift from 60 to 62 kDa. This event began by 15 min post-injection and was maximal by 45 min. Alkaline phosphatase treatment revealed this modification was caused by a change in occludin phosphorylation. In addition, the quantity of extracted occludin increased 2-fold in the same time frame. The phosphorylation and increased extraction of occludin was recapitulated in retinal endothelial cells in culture after VEGF stimulation. The data presented herein are the first demonstration of a change in the phosphorylation of this transmembrane protein under conditions of increased endothelial permeability. In addition, intra-ocular injection of VEGF also caused tyrosine phosphorylation of ZO-1 as early as 15 min and increased phosphorylation 4-fold after 90 min. In conclusion, VEGF rapidly increases occludin phosphorylation as well as the tyrosine phosphorylation of ZO-1. Phosphorylation of occludin and ZO-1 likely contribute to regulated endothelial paracellular permeability.

Vascular endothelial growth factor (VEGF) is an endothelial cell mitogen and permeability factor that is potently angiogenic in vivo. We report here studies that suggest that VEGF potentiates angiogenesis in vivo and prolongs the survival of human dermal microvascular endothelial cells (HDMECs) in vitro by inducing expression of the anti-apoptotic protein Bcl-2. Growth-factor-enriched and serum-deficient cultures of HDMECs grown on collagen type I gels with VEGF exhibited a 4-fold and a 1.6-fold reduction, respectively, in the proportion of apoptotic cells. Enhanced HDMEC survival was associated with a dose-dependent increase in Bcl-2 expression and a decrease in the expression of the processed forms of the cysteine protease caspase-3. Cultures of HDMECs transduced with and overexpressing Bcl-2 and deprived of growth factors showed enhanced protection from apoptosis and exhibited a twofold increase in cell number and a fourfold increase in the number of capillary-like sprouts. HDMECs overexpressing Bcl-2 when incorporated into polylactic acid sponges and implanted into SCID mice exhibited a sustained fivefold increase in the number of microvessels and a fourfold decrease in the number of apoptotic cells when examined 7 and 14 days later. These results suggest that the angiogenic activity attributed to VEGF may be due in part to its ability to enhance endothelial cell survival by inducing expression of Bcl-2.

Each year, more than half a million women are diagnosed with cervical cancer and the disease results in over 300 000 deaths worldwide. High-risk subtypes of the human papilloma virus (HPV) are the cause of the disease in most cases. The disease is largely preventable. Approximately 90% of cervical cancers occur in low-income and middle-income countries that lack organised screening and HPV vaccination programmes. In high-income countries, cervical cancer incidence and mortality have more than halved over the past 30 years since the introduction of formal screening programmes. Treatment depends on disease extent at diagnosis and locally available resources, and might involve radical hysterectomy or chemoradiation, or a combination of both. Conservative, fertility-preserving surgical procedures have become standard of care for women with low-risk, early-stage disease. Advances in radiotherapy technology, such as intensity-modulated radiotherapy, have resulted in less treatment-related toxicity for women with locally-advanced disease. For women with metastatic or recurrent disease, the overall prognosis remains poor; nevertheless, the incorporation of the anti-VEGF agent bevacizumab has been able to extend overall survival beyond 12 months. Preliminary results of novel immunotherapeutic approaches, similarly to other solid tumours, have shown promising results so far.

Ischemia often precedes neovascularization. In ocular neovascularization, such as occurs in diabetic retinopathy, a diffusible angiogenic factor has been postulated to be produced by ischemic retina and to lead to neovascularization of the retina, optic nerve, or iris. However, no angiogenic factor has been conclusively identified that satisfies this hypothesis. Vascular endothelial growth factor/vascular permeability factor, hereafter referred to as VEGF, is a likely candidate for an ocular angiogenic factor because it is a secreted mitogen, specific for endothelial cells, and is upregulated by hypoxia. We investigated the association of VEGF with the development of experimental iris neovascularization in the cynomolgus monkey. Following the production of retinal ischemia by laser occlusion of all branch retinal veins, VEGF was increased in the aqueous fluid, and the aqueous VEGF levels changed synchronously and proportionally with the severity of iris neovascularization. Northern analysis and in situ hybridization revealed that VEGF messenger RNA is upregulated in the ischemic retina. These observations support the hypothesis that ocular neovascularization is regulated by a diffusible factor and identify VEGF as a likely candidate for a retina-derived vascular permeability and angiogenesis factor in vivo.

BACKGROUND

When the myocardium is deprived of blood, a process of ischemia, infarction, and myocardial remodeling is initiated. Hypoxia-inducible factor 1 (HIF-1) is a transcriptional activator of vascular endothelial growth factor (VEGF) and is critical for initiating early cellular responses to hypoxia. We investigated the temporal and spatial patterns of expression of the alpha subunit of HIF-1 (HIF-1alpha) and VEGF in specimens of human heart tissue to elucidate the early molecular responses to myocardial hypoxia.

METHODS

Ventricular-biopsy specimens from 37 patients undergoing coronary bypass surgery were collected. The specimens were examined by microscopy for evidence of ischemia, evolving infarction, or a normal histologic appearance. The specimens were also analyzed with the reverse-transcriptase polymerase chain reaction for HIF-1alpha and VEGF messenger RNA (mRNA) expression and by immunohistochemical analysis for the location of the HIF-1alpha and VEGF proteins.

RESULTS

HIF-1alpha mRNA was detected in myocardial specimens with pathological evidence of acute ischemia (onset, <48 hours before surgery) or early infarction (onset, <24 hours before surgery). In contrast, VEGF transcripts were seen in specimens with evidence of acute ischemia or evolving infarction (onset, 24 to 120 hours before surgery). Patients with normal ventricles or evidence of infarction in the distant past had no detectable levels of either VEGF mRNA or HIF-1alpha mRNA. HIF-1alpha immunoreactivity was detected in the nuclei of myocytes and endothelial cells, whereas VEGF immunoreactivity was found in the cytoplasm of endothelial cells lining capillaries and arterioles.

CONCLUSIONS

An increase in the level of HIF-1alpha is an early response to myocardial ischemia or infarction. This response defines, at a molecular level, one of the first adaptations of human myocardium to a deprivation of blood. HIF-1alpha is a useful temporal marker of acutely jeopardized myocardium.

Metastasis remains one of the major challenges before hepatocellular carcinoma (HCC) is finally conquered. This paper summarized a decade's studies on HCC metastasis at the Liver Cancer Institute of Fudan University. We have established a stepwise metastatic human HCC model system, which included a metastatic HCC model in nude mice (LCI-D20), a HCC cell line with high metastatic potential (MHCC97), a relatively low metastatic potential cell clone (MHCC97L) and several stepwise high metastatic potential cell clones (MHCC97H, HCCLM3, and HCCLM6) from their parent MHCC97 cell. Endeavors have been made for searching human HCC metastasis-related chromosomes/proteins/genes. Monogene-based studies revealed that HCC invasion/metastasis was similar to that of other solid tumors, and the biological characteristics of small HCC were only slightly better than that of large HCC. Using comparative genomic hybridization (CGH), fluorescence in situ hybridization (FISH), genotyping, cDNA microarray, and 2-dimensional gel electrophoresis, we obtained some interesting results. In particular, in collaboration with the National Institute of Health (NIH) in the United States, we generated a molecular signature that can classify metastatic HCC patients, identified osteopontin as a lead gene in the signature, and found that genes favoring metastasis progression were initiated in the primary tumors. We also found that chromosome 8p deletion, particularly in the region of 8p23, was associated with HCC metastasis. Cytokeratin 19 was identified as one of the proteins, which was found in MHCC97H, but not in MHCC97L cells. Experimental interventions using the high metastatic nude mice model have provided clues for the prevention of HCC metastasis. Translation from workbench to bedside demonstrated that serum VEGF, microvessel density, and p53 scoring may be of value for the prediction of postoperative metastatic recurrence. Interferon alpha proved effective for the prevention of recurrence both experimentally and clinically. In conclusion, HCC metastasis that probably initiated in the primary tumor is a multigene-involved, multistep, and changing process. The further elucidation of the mechanism underlying HCC metastasis will provide a more solid basis for the prediction and prevention of the metastatic recurrence of HCC.

Chuvash polycythemia is an autosomal recessive disorder that is endemic to the mid-Volga River region. We previously mapped the locus associated with Chuvash polycythemia to chromosome 3p25. The gene associated with von Hippel-Lindau syndrome, VHL, maps to this region, and homozygosity with respect to a C->>T missense mutation in VHL, causing an arginine-to-tryptophan change at amino-acid residue 200 (Arg200Trp), was identified in all individuals affected with Chuvash polycythemia. The protein VHL modulates the ubiquitination and subsequent destruction of hypoxia-inducible factor 1, subunit alpha (HIF1alpha). Our data indicate that the Arg200Trp substitution impairs the interaction of VHL with HIF1alpha, reducing the rate of degradation of HIF1alpha and resulting in increased expression of downstream target genes including EPO (encoding erythropoietin), SLC2A1 (also known as GLUT1, encoding solute carrier family 2 (facilitated glucose transporter), member 1), TF (encoding transferrin), TFRC (encoding transferrin receptor (p90, CD71)) and VEGF (encoding vascular endothelial growth factor).

Expression of vascular endothelial growth factor (VEGF), a key angiogenic protein, has been linked with pancreatic cancer progression. However, the molecular basis for VEGF overexpression remains unclear. Immunohistochemical studies have indicated that VEGF overexpression coincides with elevated Stat3 activation in human pancreatic cancer specimens. In our study, more than 80% of the human pancreatic cancer cell lines used exhibited constitutively activated Stat3, with Stat3 activation correlated with the VEGF expression level. Blockade of activated Stat3 via ectopic expression of dominant-negative Stat3 significantly suppressed VEGF expression, angiogenesis, tumor growth, and metastasis in vivo. Furthermore, constitutively activated Stat3 directly activated the VEGF promoter, whereas dominant-negative Stat3 inhibited the VEGF promoter. A putative Stat3-responsive element on the VEGF promoter was identified using a protein-DNA binding assay and confirmed using a promoter mutagenesis assay. These results indicate that Stat3 directly regulates VEGF expression and hence angiogenesis, growth, and metastasis of human pancreatic cancer, suggesting that Stat3 signaling may be targeted for treatment of pancreatic cancer.

OBJECTIVE

Vascular endothelial growth factor (VEGF) is a potent and specific angiogenic growth factor, in vitro and in vivo, that may be associated with the development of intraocular neovascularization. In the current study, the authors analyze the expression of VEGF in subfoveal fibrovascular membranes from patients with age-related macular degeneration.

METHODS

Surgically removed subfoveal fibrovascular membranes from 18 eyes were analyzed for the expression of VEGF mRNA and protein using in situ hybridization and immunohistochemistry, respectively.

RESULTS

Most specimens expressed both VEGF mRNA and protein. The VEGF mRNA expression was particularly high in areas with a marked inflammatory response, in which the expression was concentrated to cells resembling fibroblasts and to surrounding inflammatory cells. VEGF protein expression was seen in fibrovascular parts of the membranes and was predominantly localized to the cytoplasm of fibroblastlike cells. In some of these membranes, strong VEGF protein immunoreactivity also was concentrated to extracellular matrix foci within the fibrovascular stroma.

CONCLUSIONS

Results indicate that VEGF may be of pathogenetic importance for the development of the choroidal neovascularization (age-related macular degeneration) and also may implicate a role of fibroblasts of presumable choroidal origin in this process.

BACKGROUND

Angiogenesis is an important therapeutic target in colorectal carcinoma. Ramucirumab is a human IgG-1 monoclonal antibody that targets the extracellular domain of VEGF receptor 2. We assessed the efficacy and safety of ramucirumab versus placebo in combination with second-line FOLFIRI (leucovorin, fluorouracil, and irinotecan) for metastatic colorectal cancer in patients with disease progression during or after first-line therapy with bevacizumab, oxaliplatin, and a fluoropyrimidine.

METHODS

Between Dec 14, 2010, and Aug 23, 2013, we enrolled patients into the multicentre, randomised, double-blind, phase 3 RAISE trial. Eligible patients had disease progression during or within 6 months of the last dose of first-line therapy. Patients were randomised (1:1) via a centralised, interactive voice-response system to receive 8 mg/kg intravenous ramucirumab plus FOLFIRI or matching placebo plus FOLFIRI every 2 weeks until disease progression, unacceptable toxic effects, or death. Randomisation was stratified by region, KRAS mutation status, and time to disease progression after starting first-line treatment. The primary endpoint was overall survival in the intention-to-treat population. This study is registered with ClinicalTrials.gov, number NCT01183780.ld

RESULTS

We enrolled 1072 patients (536 in each group). Median overall survival was 13·3 months (95% CI 12·4-14·5) for patients in the ramucirumab group versus 11·7 months (10·8-12·7) for the placebo group (hazard ratio 0·844 95% CI 0·730-0·976; log-rank p=0·0219). Survival benefit was consistent across subgroups of patients who received ramucirumab plus FOLFIRI. Grade 3 or worse adverse events seen in more than 5% of patients were neutropenia (203 [38%] of 529 patients in the ramucirumab group vs 123 [23%] of 528 in the placebo group, with febrile neutropenia incidence of 18 [3%] vs 13 [2%]), hypertension (59 [11%] vs 15 [3%]), diarrhoea (57 [11%] vs 51 [10%]), and fatigue (61 [12%] vs 41 [8%]).

CONCLUSIONS

Ramucirumab plus FOLFIRI significantly improved overall survival compared with placebo plus FOLFIRI as second-line treatment for patients with metastatic colorectal carcinoma. No unexpected adverse events were identified and toxic effects were manageable.

BACKGROUND

Eli Lilly.

Immune escape is a prerequisite for tumor development. To avoid the immune system, tumors develop different mechanisms, including T cell exhaustion, which is characterized by expression of immune inhibitory receptors, such as PD-1, CTLA-4, Tim-3, and a progressive loss of function. The recent development of therapies targeting PD-1 and CTLA-4 have raised great interest since they induced long-lasting objective responses in patients suffering from advanced metastatic tumors. However, the regulation of PD-1 expression, and thereby of exhaustion, is unclear. VEGF-A, a proangiogenic molecule produced by the tumors, plays a key role in the development of an immunosuppressive microenvironment. We report in the present work that VEGF-A produced in the tumor microenvironment enhances expression of PD-1 and other inhibitory checkpoints involved in CD8(+) T cell exhaustion, which could be reverted by anti-angiogenic agents targeting VEGF-A-VEGFR. In view of these results, association of anti-angiogenic molecules with immunomodulators of inhibitory checkpoints may be of particular interest in VEGF-A-producing tumors.

Endothelial permeability depends on the integrity of intercellular junctions as well as actomyosin-based cell contractility. Rho GTPases have been implicated in signalling by many vasoactive substances including thrombin, tumour necrosis factor alpha (TNF-alpha), bradykinin, histamine, lysophosphatidic acid (LPA), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF). Two Rho family GTPases, Rho and Rac, have emerged as key regulators acting antagonistically to regulate endothelial barrier function: Rho increases actomyosin contractility, which facilitates breakdown of intercellular junctions, whereas Rac stabilizes endothelial junctions and counteracts the effects of Rho. In this review, we present evidence for the opposing effects of these two regulatory proteins and discuss links between them and other key signalling molecules such as cyclic AMP (cAMP), cyclic GMP (cGMP), phosphatidylinositide 3-kinases (PI3Ks), mitogen-activated protein kinases (MAPKs), and protein kinases C (PKCs). We also discuss strategies for targeting Rho GTPase signalling in therapies for diseases involving altered endothelial permeability.

c-Myc promotes cell growth and transformation by ill-defined mechanisms. c-myc(-/-) mice die by embryonic day 10.5 (E10.5) with defects in growth and in cardiac and neural development. Here we report that the lethality of c-myc(-/-) embryos is also associated with profound defects in vasculogenesis and primitive erythropoiesis. Furthermore, c-myc(-/-) embryonic stem (ES) and yolk sac cells are compromised in their differentiative and growth potential. These defects are intrinsic to c-Myc, and are in part associated with a requirement for c-Myc for the expression of vascular endothelial growth factor (VEGF), as VEGF can partially rescue these defects. However, c-Myc is also required for the proper expression of other angiogenic factors in ES and yolk sac cells, including angiopoietin-2, and the angiogenic inhibitors thrombospondin-1 and angiopoietin-1. Finally, c-myc(-/-) ES cells are dramatically impaired in their ability to form tumors in immune-compromised mice, and the small tumors that sometimes develop are poorly vascularized. Therefore, c-Myc function is also necessary for the angiogenic switch that is indispensable for the progression and metastasis of tumors. These findings support the model wherein c-Myc promotes cell growth and transformation, as well as vascular and hematopoietic development, by functioning as a master regulator of angiogenic factors.

Genetically engineered mice (Myf5nLacZ/+, Myf5GFP-P/+) allowing direct muscle satellite cell (SC) visualization indicate that, in addition to being located beneath myofiber basal laminae, SCs are strikingly close to capillaries. After GFP(+) bone marrow transplantation, blood-borne cells occupying SC niches previously depleted by irradiation were similarly detected near vessels, thereby corroborating the anatomical stability of juxtavascular SC niches. Bromodeoxyuridine pulse-chase experiments also localize quiescent and less quiescent SCs near vessels. SCs, and to a lesser extent myonuclei, were nonrandomly associated with capillaries in humans. Significantly, they were correlated with capillarization of myofibers, regardless to their type, in normal muscle. They also varied in paradigmatic physiological and pathological situations associated with variations of capillary density, including amyopathic dermatomyositis, a unique condition in which muscle capillary loss occurs without myofiber damage, and in athletes in whom capillaries increase in number. Endothelial cell (EC) cultures specifically enhanced SC growth, through IGF-1, HGF, bFGF, PDGF-BB, and VEGF, and, accordingly, cycling SCs remained mainly juxtavascular. Conversely, differentiating myogenic cells were both proangiogenic in vitro and spatiotemporally associated with neoangiogenesis in muscular dystrophy. Thus, SCs are largely juxtavascular and reciprocally interact with ECs during differentiation to support angio-myogenesis.