BACKGROUND

The expression of carcino-embryonic antigen by colorectal cancer is an example of oncogenic activation of embryonic gene expression. Hypothesizing that oncogenesis-recapitulating-ontogenesis may represent a broad programmatic commitment, we compared gene expression patterns of human colorectal cancers (CRCs) and mouse colon tumor models to those of mouse colon development embryonic days 13.5-18.5.

RESULTS

We report here that 39 colon tumors from four independent mouse models and 100 human CRCs encompassing all clinical stages shared a striking recapitulation of embryonic colon gene expression. Compared to normal adult colon, all mouse and human tumors over-expressed a large cluster of genes highly enriched for functional association to the control of cell cycle progression, proliferation, and migration, including those encoding MYC, AKT2, PLK1 and SPARC. Mouse tumors positive for nuclear beta-catenin shifted the shared embryonic pattern to that of early development. Human and mouse tumors differed from normal embryonic colon by their loss of expression modules enriched for tumor suppressors (EDNRB, HSPE, KIT and LSP1). Human CRC adenocarcinomas lost an additional suppressor module (IGFBP4, MAP4K1, PDGFRA, STAB1 and WNT4). Many human tumor samples also gained expression of a coordinately regulated module associated with advanced malignancy (ABCC1, FOXO3A, LIF, PIK3R1, PRNP, TNC, TIMP3 and VEGF).

CONCLUSIONS

Cross-species, developmental, and multi-model gene expression patterning comparisons provide an integrated and versatile framework for definition of transcriptional programs associated with oncogenesis. This approach also provides a general method for identifying pattern-specific biomarkers and therapeutic targets. This delineation and categorization of developmental and non-developmental activator and suppressor gene modules can thus facilitate the formulation of sophisticated hypotheses to evaluate potential synergistic effects of targeting within- and between-modules for next-generation combinatorial therapeutics and improved mouse models.

Little is known about the formation of niches, local micro-environments required for stem-cell maintenance. Here we develop an in vivo assay for adult haematopoietic stem-cell (HSC) niche formation. With this assay, we identified a population of progenitor cells with surface markers CD45(-)Tie2(-)alpha(V)(+)CD105(+)Thy1.1(-) (CD105(+)Thy1(-)) that, when sorted from 15.5 days post-coitum fetal bones and transplanted under the adult mouse kidney capsule, could recruit host-derived blood vessels, produce donor-derived ectopic bones through a cartilage intermediate and generate a marrow cavity populated by host-derived long-term reconstituting HSC (LT-HSC). In contrast, CD45(-)Tie2(-)alpha(V)(+)CD105(+)Thy1(+) (CD105(+)Thy1(+)) fetal bone progenitors form bone that does not contain a marrow cavity. Suppressing expression of factors involved in endochondral ossification, such as osterix and vascular endothelial growth factor (VEGF), inhibited niche generation. CD105(+)Thy1(-) progenitor populations derived from regions of the fetal mandible or calvaria that do not undergo endochondral ossification formed only bone without marrow in our assay. Collectively, our data implicate endochondral ossification, bone formation that proceeds through a cartilage intermediate, as a requirement for adult HSC niche formation.

The pathway involving the tumor suppressor gene TP53 can regulate tumor angiogenesis by unclear mechanisms. Here we show that p53 regulates hypoxic signaling through the transcriptional regulation of microRNA-107 (miR-107). We found that miR-107 is a microRNA expressed by human colon cancer specimens and regulated by p53. miR-107 decreases hypoxia signaling by suppressing expression of hypoxia inducible factor-1beta (HIF-1beta). Knockdown of endogenous miR-107 enhances HIF-1beta expression and hypoxic signaling in human colon cancer cells. Conversely, overexpression of miR-107 inhibits HIF-1beta expression and hypoxic signaling. Furthermore, overexpression of miR-107 in tumor cells suppresses tumor angiogenesis, tumor growth, and tumor VEGF expression in mice. Finally, in human colon cancer specimens, expression of miR-107 is inversely associated with expression of HIF-1beta. Taken together these data suggest that miR-107 can mediate p53 regulation of hypoxic signaling and tumor angiogenesis.

OBJECTIVE

A randomized, phase III, placebo-controlled, partially blinded clinical trial (REGAL [Recent in in Glioblastoma Alone and With Lomustine]) was conducted to determine the efficacy of cediranib, an oral pan-vascular endothelial growth factor (VEGF) receptor tyrosine kinase inhibitor, either as monotherapy or in combination with lomustine versus lomustine in patients with recurrent glioblastoma.

METHODS

Patients (N = 325) with recurrent glioblastoma who previously received radiation and temozolomide were randomly assigned 2:2:1 to receive (1) cediranib (30 mg) monotherapy; (2) cediranib (20 mg) plus lomustine (110 mg/m(2)); (3) lomustine (110 mg/m(2)) plus a placebo. The primary end point was progression-free survival based on blinded, independent radiographic assessment of postcontrast T1-weighted and noncontrast T2-weighted magnetic resonance imaging (MRI) brain scans.

RESULTS

The primary end point of progression-free survival (PFS) was not significantly different for either cediranib alone (hazard ratio [HR] = 1.05; 95% CI, 0.74 to 1.50; two-sided P = .90) or cediranib in combination with lomustine (HR = 0.76; 95% CI, 0.53 to 1.08; two-sided P = .16) versus lomustine based on independent or local review of postcontrast T1-weighted MRI.

CONCLUSIONS

This study did not meet its primary end point of PFS prolongation with cediranib either as monotherapy or in combination with lomustine versus lomustine in patients with recurrent glioblastoma, although cediranib showed evidence of clinical activity on some secondary end points including time to deterioration in neurologic status and corticosteroid-sparing effects.

The murine VEGF gene is alternatively transcribed to yield the VEGF(120), VEGF(164), and VEGF(188) isoforms, which differ in their potential to bind to heparan sulfate and neuropilin-1 and to stimulate endothelial growth. Here, their role in retinal vascular development was studied in mice selectively expressing single isoforms. VEGF(164/164) mice were normal, healthy, and had normal retinal angiogenesis. In contrast, VEGF(120/120) mice exhibited severe defects in vascular outgrowth and patterning, whereas VEGF(188/188) mice displayed normal venular outgrowth but impaired arterial development. It is noteworthy that neuropilin-1, a receptor for VEGF(164), was predominantly expressed in retinal arterioles. These findings reveal distinct roles of the various VEGF isoforms in vascular patterning and arterial development in the retina.

Local, controlled induction of angiogenesis remains a challenge that limits tissue engineering approaches to replace or restore diseased tissues. We present a new class of bioactive synthetic hydrogel matrices based on poly(ethylene glycol) (PEG) and synthetic peptides that exploits the activity of vascular endothelial growth factor (VEGF) alongside the base matrix functionality for cellular ingrowth, that is, induction of cell adhesion by pendant RGD-containing peptides and provision of cell-mediated remodeling by cross-linking matrix metalloproteinase substrate peptides. By using a Michael-type addition reaction, we incorporated variants of VEGFVEGFVEGF was preserved and was critical for in vitro endothelial cell survival and migration within the matrix environment. Consistent with a scheme of locally restricted availability of VEGF, grafting of these VEGF-modified hydrogel matrices atop the chick chorioallontoic membrane evoked strong new blood vessel formation precisely at the area of graft-membrane contact. When implanted subcutaneously in rats, these VEGF-containing matrices were completely remodeled into native, vascularized tissue. This type of synthetic, biointeractive matrix with integrated angiogenic growth factor activity, presented and released only upon local cellular demand, could become highly useful in a number of clinical healing applications of local therapeutic angiogenesis.

Vascular endothelial cells are ordinarily quiescent in adult humans and divide less than once per decade. When tumors reach a size of about 0.2-2.0mm in diameter, they become hypoxic and limited in size in the absence of angiogenesis. There are about 30 endogenous pro-angiogenic factors and about 30 endogenous anti-angiogenic factors. In order to increase in size, tumors undergo an angiogenic switch where the action of pro-angiogenic factors predominates, resulting in angiogenesis and tumor progression. One mechanism for driving angiogenesis results from the increased production of vascular endothelial growth factor (VEGF) following up-regulation of the hypoxia-inducible transcription factor. The human VEGF family consists of VEGF (VEGF-A), VEGF-B, VEGF-C, VEGF-D, and placental growth factor (PlGF). The VEGF family of receptors consists of three protein-tyrosine kinases and two non-protein kinase receptors (neuropilin-1 and -2). Owing to the importance of angiogenesis in tumor progression, inhibition of VEGF signaling represents an attractive cancer treatment.

The measurement of circulating vascular endothelial growth factor (VEGF) levels as a prognostic factor will gain increasing relevance in the diagnosis and evaluation of treatment in cancer patients. Angiogenesis is an absolute requirement in tumour growth and metastatic disease. In the present study data are presented which indicate that circulating VEGF mainly resides in peripheral blood cells. In 15 healthy volunteers we demonstrated that approximately 34% of the circulating VEGF resides in platelets and approximately 11% in patients with cancer ( n = 4). An important part namely 58% in healthy volunteers and 69% in patients with cancer of the total circulating VEGF is contained in granulocytes, particular in the neutrophils, as confirmed by fluorescence-activated cell sorting (FACS). Also an increased VEGF level per granulocyte is found in patients with cancer (77 microg VEGF/l) compared with the healthy volunteers (164 microg VEGF/l). In contrast only 2% was present in plasma. The biological significance of platelet- or granulocyte-derived VEGF is not yet known. Liberation of VEGF from these compartments could well be of importance for tumour angiogenesis. Therefore, future studies on the clinical value of circulating VEGF as a prognostic factor in cancer patients should include measurements of VEGF in peripheral blood cells.

Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF, VEGF-A) is a multifunctional cytokine with important roles in pathological angiogenesis. Using an adenoviral vector engineered to express murine VEGF-A(164), we previously investigated the steps and mechanisms by which this cytokine induced the formation of new blood vessels in adult immunodeficient mice and demonstrated that the newly formed blood vessels closely resembled those found in VEGF-A-expressing tumors. We now report that, in addition to inducing angiogenesis, VEGF-A(164) also induces a strong lymphangiogenic response. This finding was unanticipated because lymphangiogenesis has been thought to be mediated by other members of the VPF/VEGF family, namely, VEGF-C and VEGF-D. The new "giant" lymphatics generated by VEGF-A(164) were structurally and functionally abnormal: greatly enlarged with incompetent valves, sluggish flow, and delayed lymph clearance. They closely resembled the large lymphatics found in lymphangiomas/lymphatic malformations, perhaps implicating VEGF-A in the pathogenesis of these lesions. Whereas the angiogenic response was maintained only as long as VEGF-A was expressed, giant lymphatics, once formed, became VEGF-A independent and persisted indefinitely, long after VEGF-A expression ceased. These findings raise the possibility that similar, abnormal lymphatics develop in other pathologies in which VEGF-A is overexpressed, e.g., malignant tumors and chronic inflammation.

Hepatocellular carcinoma (HCC), the most common primary liver tumor, is notoriously resistant to systemic therapies, and often recurs even after aggressive local therapies. HCCs rely on the formation of new blood vessels for growth, and VEGF is critical in this process. A hallmark of new vessel formation in tumors is their structural and functional abnormality. This leads to an abnormal tumor microenvironment characterized by low oxygen tension. The liver is perfused by both arterial and venous blood and the resulting abnormal microenvironment selects for more-aggressive malignancies. Anti-VEGF therapy with sorafenib was the first systemic therapy to demonstrate improved survival in patients with advanced-stage HCC. This important development in the treatment of HCC raises hope as well as critical questions on the future development of targeted agents including other antiangiogenic agents, which hold promise to further increase survival in this aggressive disease.

The involvement of immune mechanisms in tumor angiogenesis is unclear. Here we describe a new mechanism of tumor vasculogenesis mediated by dendritic cell (DC) precursors through the cooperation of beta-defensins and vascular endothelial growth factor-A (Vegf-A). Expression of mouse beta-defensin-29 recruited DC precursors to tumors and enhanced tumor vascularization and growth in the presence of increased Vegf-A expression. A new leukocyte population expressing DC and endothelial markers was uncovered in mouse and human ovarian carcinomas coexpressing Vegf-A and beta-defensins. Tumor-infiltrating DCs migrated to tumor vessels and independently assembled neovasculature in vivo. Bone marrow-derived DCs underwent endothelial-like differentiation ex vivo, migrated to blood vessels and promoted the growth of tumors expressing high levels of Vegf-A. We show that beta-defensins and Vegf-A cooperate to promote tumor vasculogenesis by carrying out distinct tasks: beta-defensins chemoattract DC precursors through CCR6, whereas Vegf-A primarily induces their endothelial-like specialization and migration to vessels, which is mediated by Vegf receptor-2.

Use of long-term constitutive expression of VEGF for therapeutic angiogenesis may be limited by the growth of abnormal blood vessels and hemangiomas. We investigated the relationship between VEGF dosage and the morphology and function of newly formed blood vessels by implanting retrovirally transduced myoblasts that constitutively express VEGFVEGF dosage by decreasing the total number of VEGF myoblasts implanted did not prevent vascular abnormalities. However, when clonal populations of myoblasts homogeneously expressing different levels of VEGF were implanted, a threshold between normal and aberrant angiogenesis was found. Clonal myoblasts that expressed low to medium levels of VEGF induced growth of stable, pericyte-coated capillaries of uniform size that were not leaky and became VEGF independent, as shown by treatment with the potent VEGF blocker VEGF-TrapR1R2. In contrast, clones that expressed high levels of VEGF induced hemangiomas. Remarkably, when different clonal populations were mixed, even a small proportion of cells with high production of VEGF was sufficient to cause hemangioma growth. These results show for the first time to our knowledge that the key determinant of whether VEGF-induced angiogenesis is normal or aberrant is the microenvironmental amount of growth factor secreted, rather than the overall dose. Long-term continuous delivery of VEGF, when maintained below a threshold microenvironmental level, can lead to normal angiogenesis without other exogenous growth factors.

Hormone refractory metastatic prostate cancer remains an incurable disease. We found that high expression levels of the chemokine receptor CXCR4 correlated with the presence of metastatic disease in prostate cancer patients. Positive staining for CXCL12, the ligand for CXCR4, was mainly present in the tumor-associated blood vessels and basal cell hyperplasia. Subcutaneous xenografts of PC3 and 22Rv1 prostate tumors that overexpressed CXCR4 in NOD/SCID mice were two- to threefold larger in volume and weight vs. controls. Moreover, blood vessel density, functionality, invasiveness of tumors into the surrounding tissues, and metastasis to the lymph node and lung were significantly increased in these tumors. Neutralizing the interactions of CXCL12/CXCR4 in vivo with CXCR4 specific antibodies inhibited the CXCR4-dependent tumor growth and vascularization. In vitro, CXCL12 induced the proliferation and VEGF secretion but not migration of PC3 and 22Rv1 cells overexpressing CXCR4. Similar effects of CXCR4 overexpression on tumor growth in vivo were also noted in two breast cancer lines, suggesting that the observed effect of CXCR4 is not unique to prostate tumor cells. Thus high levels of the chemokine receptor CXCR4 induce a more aggressive phenotype in prostate cancer cells and identify CXCR4 as a potential therapeutic target in advanced cases of metastatic prostate cancer.

OBJECTIVE

The expansion of adipose tissue mass seen in obesity involves both hyperplasia and hypertrophy of adipocytes. However, little is known about how adipocytes, adipocyte precursors, blood vessels, and stromal cells interact with one another to achieve adipogenesis.

METHODS

We have developed a confocal microscopy-based method of three-dimensional visualization of intact living adipose tissue that enabled us to simultaneously evaluate angiogenesis and adipogenesis in db/db mice.

RESULTS

We found that adipocyte differentiation takes place within cell clusters (which we designated adipogenic/angiogenic cell clusters) that contain multiple cell types, including endothelial cells and stromal cells that express CD34 and CD68 and bind lectin. There were close spatial and temporal interrelationships between blood vessel formation and adipogenesis, and the sprouting of new blood vessels from preexisting vasculature was coupled to adipocyte differentiation. CD34(+) CD68(+) lectin-binding cells could clearly be distinguished from CD34(-) CD68(+) macrophages, which were scattered in the stroma and did not bind lectin. Adipogenic/angiogenic cell clusters can morphologically and immunohistochemically be distinguished from crown-like structures frequently seen in the late stages of adipose tissue obesity. Administration of anti-vascular endothelial growth factor (VEGF) antibodies inhibited not only angiogenesis but also the formation of adipogenic/angiogenic cell clusters, indicating that the coupling of adipogenesis and angiogenesis is essential for differentiation of adipocytes in obesity and that VEGF is a key mediator of that process.

CONCLUSIONS

Living tissue imaging techniques provide novel evidence of the dynamic interactions between differentiating adipocytes, stromal cells, and angiogenesis in living obese adipose tissue.

Vascular endothelial growth factor receptor-3 (VEGFR-3) has an essential role in the development of embryonic blood vessels; however, after midgestation its expression becomes restricted mainly to the developing lymphatic vessels. The VEGFR-3 ligand VEGF-C stimulates lymphangiogenesis in transgenic mice and in chick chorioallantoic membrane. As VEGF-C also binds VEGFR-2, which is expressed in lymphatic endothelia, it is not clear which receptors are responsible for the lymphangiogenic effects of VEGF-C. VEGF-D, which binds to the same receptors, has been reported to induce angiogenesis, but its lymphangiogenic potential is not known. In order to define the lymphangiogenic signalling pathway we have created transgenic mice overexpressing a VEGFR-3-specific mutant of VEGF-C (VEGF-C156S) or VEGF-D in epidermal keratinocytes under the keratin 14 promoter. Both transgenes induced the growth of lymphatic vessels in the skin, whereas the blood vessel architecture was not affected. Evidence was also obtained that these growth factors act in a paracrine manner in vivo. These results demonstrate that stimulation of the VEGFR-3 signal transduction pathway is sufficient to induce specifically lymphangiogenesis in vivo.

This Chapter has reviewed the literature concerning VPF/VEGF as a potent vascular permeabilizing cytokine. In accord with this important role, microvessels have been found to be hyperpermeable to plasma proteins and other circulating macromolecules at sites where VPF/VEGF and its receptors are overexpressed, i.e., in tumors, healing wounds, retinopathies, many important inflammatory conditions and in certain physiological processes, such as ovulation and corpus luteum formation. Moreover, microvascular hyperpermeability to plasma proteins was shown to have an important consequence: the laying down of a fibrin-rich extracellular matrix. This provisional matrix, in turn, favors and supports the ingrowth of fibroblasts and endothelial cells which, together, transform the provisional matrix into the mature stroma characteristic of tumors and healed wounds. Finally, we have considered the pathways by which these and other circulating macromolecules cross the endothelium of normal and VPF/VEGF-permeabilized microvessels. These pathways include VVOs and trans-endothelial openings that have been variously interpreted as inter-endothelial cell gaps or trans-endothelial cell pores. At least some trans-endothelial cell pores may arise from VVOs. In conclusion, these data provide new insights into the mechanisms of angiogenesis and stroma formation, insights which are potentially applicable to a wide variety of disease states and which may lead to identification of new targets for therapeutic intervention.

Clinical practice reveals that therapy with angiogenesis inhibitors often does not prolong survival of cancer patients for more than months, because tumors elicit evasive resistance. In this issue of Cancer Cell, two papers report that VEGF inhibitors reduce primary tumor growth but promote tumor invasiveness and metastasis. These perplexing findings help to explain resistance to these drugs but raise pertinent questions of how to best treat cancer patients with antiangiogenic medicine in the future. We discuss here how VEGF inhibitors can induce such divergent effects on primary tumor growth and metastasis.

Interaction between integrin alphavbeta3 and extracellular matrix is crucial for endothelial cells sprouting from capillaries and for angiogenesis. Furthermore, integrin-mediated outside-in signals co-operate with growth factor receptors to promote cell proliferation and motility. To determine a potential regulation of angiogenic inducer receptors by the integrin system, we investigated the interaction between alphavbeta3 integrin and tyrosine kinase vascular endothelial growth factor receptor-2 (VEGFR-2) in human endothelial cells. We report that tyrosine-phosphorylated VEGFR-2 co-immunoprecipitated with beta3 integrin subunit, but not with beta1 or beta5, from cells stimulated with VEGF-A165. VEGFR-2 phosphorylation and mitogenicity induced by VEGF-A165 were enhanced in cells plated on the alphavbeta3 ligand, vitronectin, compared with cells plated on the alpha5beta1 ligand, fibronectin or the alpha2beta1 ligand, collagen. BV4 anti-beta3 integrin mAb, which does not interfere with endothelial cell adhesion to vitronectin, reduced (i) the tyrosine phosphorylation of VEGFR-2; (ii) the activation of downstream transductor phosphoinositide 3-OH kinase; and (iii) biological effects triggered by VEGF-A165. These results indicate a new role for alphavbeta3 integrin in the activation of an in vitro angiogenic program in endothelial cells. Besides being the most important survival system for nascent vessels by regulating cell adhesion to matrix, alphavbeta3 integrin participates in the full activation of VEGFR-2 triggered by VEGF-A, which is an important angiogenic inducer in tumors, inflammation and tissue regeneration.

OBJECTIVE

To identify, localize, and determine M1/M2 polarization of epidydimal adipose tissue (eAT) macrophages (Phis) during high-fat diet (HFD)-induced obesity.

METHODS

Male C57BL/6 mice were fed an HFD (60% fat kcal) or low-fat diet (LFD) (10% fat kcal) for 8 or 12 weeks. eATMPhis (F4/80(+) cells) were characterized by in vivo fluorescent labeling, immunohistochemistry, fluorescence-activated cell sorting, and quantitative PCR.

RESULTS

Recruited interstitial macrophage galactose-type C-type lectin (MGL)1(+)/CD11c(-) and crown-like structure-associated MGL1(-)/CD11c(+) and MGL1(med)/CD11c(+) eATMPhis were identified after 8 weeks of HFD. MGL1(med)/CD11c(+) cells comprised approximately 65% of CD11c(+) eATMPhis. CD11c(+) eATMPhis expressed a mixed M1/M2 profile, with some M1 transcripts upregulated (IL-12p40 and IL-1beta), others downregulated (iNOS, caspase-1, MCP-1, and CD86), and multiple M2 and matrix remodeling transcripts upregulated (arginase-1, IL-1Ra, MMP-12, ADAM8, VEGF, and Clec-7a). At HFD week 12, each eATMPhi subtype displayed an enhanced M2 phenotype as compared with HFD week 8. CD11c(+) subtypes downregulated IL-1beta and genes mediating antigen presentation (I-a, CD80) and upregulated the M2 hallmark Ym-1 and genes promoting oxidative metabolism (PGC-1alpha) and adipogenesis (MMP-2). MGL1(med)/CD11c(+) eATMPhis upregulated additional M2 genes (IL-13, SPHK1, CD163, LYVE-1, and PPAR-alpha). MGL1(med)/CD11c(+) ATMPhis expressing elevated PGC-1alpha, PPAR-alpha, and Ym-1 transcripts were selectively enriched in eAT of obese mice fed pioglitazone for 6 days, confirming the M2 features of the MGL1(med)/CD11c(+) eATMPhi transcriptional profile and implicating PPAR activation in its elicitation.

CONCLUSIONS

These results 1) redefine the phenotypic potential of CD11c(+) eATMPhis and 2) suggest previously unappreciated phenotypic and functional commonality between murine and human ATMPhis in the development of obesity and its complications.

Extensive research within the last decade has revealed that most chronic illnesses such as cancer, cardiovascular and pulmonary diseases, neurological diseases, diabetes, and autoimmune diseases exhibit dysregulation of multiple cell signaling pathways that have been linked to inflammation. Thus mono-targeted therapies developed for the last two decades for these diseases have proven to be unsafe, ineffective and expensive. Although fruits and vegetables are regarded to have therapeutic potential against chronic illnesses, neither their active component nor the mechanism of action is well understood. Resveratrol (trans-3, 5, 4'-trihydroxystilbene), a component of grapes, berries, peanuts and other traditional medicines, is one such polyphenol that has been shown to mediate its effects through modulation of many different pathways. This stilbene has been shown to bind to numerous cell-signaling molecules such as multi drug resistance protein, topoisomerase II, aromatase, DNA polymerase, estrogen receptors, tubulin and F1-ATPase. Resveratrol has also been shown to activate various transcription factor (e.g; NFkappaB, STAT3, HIF-1alpha, beta-catenin and PPAR-gamma), suppress the expression of antiapoptotic gene products (e.g; Bcl-2, Bcl-X(L), XIAP and survivin), inhibit protein kinases (e.g; src, PI3K, JNK, and AKT), induce antioxidant enzymes (e,g; catalase, superoxide dismutase and hemoxygenase-1), suppress the expression of inflammatory biomarkers (e.g., TNF, COX-2, iNOS, and CRP), inhibit the expression of angiogenic and metastatic gene products (e.g., MMPs, VEGF, cathepsin D, and ICAM-1), and modulate cell cycle regulatory genes (e.g., p53, Rb, PTEN, cyclins and CDKs). Numerous animal studies have demonstrated that this polyphenol holds promise against numerous age-associated diseases including cancer, diabetes, Alzheimer, cardiovascular and pulmonary diseases. In view of these studies, resveratrol's prospects for use in the clinics are rapidly accelerating. Efforts are also underway to improve its activity in vivo through structural modification and reformulation. Our review describes various targets of resveratrol and their therapeutic potential.

Two distinct receptors for vascular endothelial growth factor (VEGF), the tyrosine kinase receptors Flt-1 and Flk-1/KDR, have been described. In this study we show that monocytes, in contrast to endothelium, express only the VEGF receptor Flt-1, and that this receptor specifically binds also the VEGF homolog placenta growth factor (PlGF). Both VEGF and PlGF stimulate tissue factor production and chemotaxis in monocytes at equivalent doses. In contrast, endothelial cells expressing both the Flt-1 and the Flk-1/KDR receptors produce more tissue factor upon stimulation with VEGF than after stimulation with PlGF. Neutralizing antibodies to the KDR receptor reduce the VEGF-stimulated tissue factor induction in endothelial cells to levels obtained by stimulation with PlGF alone, but do not affect PlGF-induced tissue factor induction in endothelial cells nor the VEGF-dependent tissue factor production in monocytes. These findings strongly suggest Flt-1 as a functional receptor for VEGF and PlGF in monocytes and endothelial cells and identify this receptor as a mediator of monocyte recruitment and procoagulant activity.

Early imaging or blood biomarkers of tumor response are desperately needed to customize antiangiogenic therapy for cancer patients. Anti-vascular endothelial growth factor (VEGF) therapy can "normalize" brain tumor vasculature by decreasing vessel diameter and permeability, and thinning the abnormally thick basement membrane. We hypothesized that the extent of vascular normalization will be predictive of outcome of anti-VEGF therapy in glioblastoma. We used advanced magnetic resonance imaging methods to monitor vascular parameters and treatment response in 31 recurrent glioblastoma patients enrolled in a phase II trial of cediranib, an oral pan-VEGF receptor tyrosine kinase inhibitor. We evaluated the correlation between clinical outcome and magnetic resonance imaging-measured changes in vascular permeability/flow (i.e., K(trans)) and in microvessel volume, and the change of circulating collagen IV levels, all after a single dose of cediranib. Here, we show that evaluation of biomarkers as early as after one day of anti-VEGF therapy with cediranib is predictive of response in patients with recurrent glioblastoma. Changes in K(trans), microvessel volume, and circulating collagen IV correlated with duration of overall survival and/or progression-free survival (P < 0.05). When we combined these three parameters into a "vascular normalization index," we found that it closely associated with overall survival (rho = 0.54; P = 0.004) and progression-free survival (rho = 0.6; P = 0.001). The vascular normalization index described here should be validated in randomized clinical trials.

CYR61 (CCN1) is a member of the CCN family of secreted matricellular proteins that includes connective tissue growth factor (CCN2), NOV (CCN3), WISP-1 (CCN4), WISP-2 (CCN5), and WISP-3 (CCN6). First identified as the product of a growth factor-inducible immediate-early gene, CYR61 is an extracellular matrix-associated angiogenic inducer that functions as a ligand of integrin receptors to promote cell adhesion, migration, and proliferation. Aberrant expression of Cyr61 is associated with breast cancer, wound healing, and vascular diseases such as atherosclerosis and restenosis. To understand the functions of CYR61 during development, we have disrupted the Cyr61 gene in mice. We show here that Cyr61-null mice suffer embryonic death: approximately 30% succumbed to a failure in chorioallantoic fusion, and the reminder perished due to placental vascular insufficiency and compromised vessel integrity. These findings establish CYR61 as a novel and essential regulator of vascular development. CYR61 deficiency results in a specific defect in vessel bifurcation (nonsprouting angiogenesis) at the chorioallantoic junction, leading to an undervascularization of the placenta without affecting differentiation of the labyrinthine syncytiotrophoblasts. This unique phenotype is correlated with impaired Vegf-C expression in the allantoic mesoderm, suggesting that CYR61-regulated expression of Vegf-C plays a role in vessel bifurcation. The genetic and molecular basis of vessel bifurcation is presently unknown, and these findings provide new insight into this aspect of angiogenesis.