We are creating synthetic pharmaceuticals with angiogenic activity and potential to promote vascular invasion. We previously demonstrated that one of these molecules, phthalimide neovascular factor 1 (PNF1), significantly expands microvascular networks in vivo following sustained release from poly(lactic-co-glycolic acid) (PLAGA) films. In addition, to probe PNF1 mode-of-action, we recently applied a novel pathway-based compendium analysis to a multi-timepoint, controlled microarray dataset of PNF1-treated (versus control) human microvascular endothelial cells (HMVECs), and we identified induction of tumor necrosis factor-alpha (TNF-α) and, subsequently, transforming growth factor-beta (TGF-β) signaling networks by PNF1. Here we validate this microarray data-set with quantitative real-time polymerase chain reaction (RT-PCR) analysis. Subsequently, we probe this dataset and identify three specific TGF-β-induced genes with regulation by PNF1 conserved over multiple timepoints—amyloid beta (A4) precursor protein (APP), early growth response 1 (EGR-1), and matrix metalloproteinase 14 (MMP14 or MT1-MMP)—that are also implicated in angiogenesis. We further focus on MMP14 given its unique role in angiogenesis, and we validate MT1-MMP modulation by PNF1 with an in vitro fluorescence assay that demonstrates the direct effects that PNF1 exerts on functional metalloproteinase activity. We also utilize endothelial cord formation in collagen gels to show that PNF1-induced stimulation of endothelial cord network formation in vitro is in some way MT1-MMP-dependent. Ultimately, this new network analysis of our transcriptional footprint characterizing PNF1 activity 1–48 h post-supplementation in HMVECs coupled with corresponding validating experiments suggests a key set of a few specific targets that are involved in PNF1 mode-of-action and important for successful promotion of the neovascularization that we have observed by the drug in vivo.
Network analysis; transcriptional profiling; angiogenesis; matrix metalloproteinase; small molecule; drug discovery
Phthalimide neovascular factor (PNF1, formerly SC-3-149) is a potent stimulator of proangiogenic signaling pathways in endothelial cells. In this study, we evaluated the in vivo effects of sustained PNF1 release to promote ingrowth and expansion of microvascular networks surrounding biomaterial implants. The dorsal skinfold window chamber was used to evaluate the structural remodeling response of the local microvasculature. PNF1 was released from poly(lactic-co-glycolic acid) (PLAGA) films, and a transport model was utilized to predict PNF1 penetration into the surrounding tissue. PNF1 significantly expanded microvascular networks within a 2 mm radius from implants after 3 and 7 days by increasing microvessel length density and lumenal diameter of local arterioles and venules. Staining of histological sections with CD11b showed enhanced recruitment of circulating white blood cells, including monocytes, which are critical for the process of vessel enlargement through arteriogenesis. As PNF1 has been shown to modulate MT1-MMP, a facilitator of CCL2 dependent leukocyte transmigration, aspects of window chamber experiments were repeated in CCR2−/− (CCL2 receptor) mouse chimeras to more fully explore the critical nature of monocyte recruitment on the therapeutic benefits of PNF1 function in vivo.
Small molecule delivery; Controlled release; Microvascular remodeling; Monocyte recruitment
Motivation: Computational techniques have been applied to experimental datasets to identify drug mode-of-action. A shortcoming of existing approaches is the requirement of large reference databases of compound expression profiles. Here, we developed a new pathway-based compendium analysis that couples multi-timepoint, controlled microarray data for a single compound with systems-based network analysis to elucidate drug mechanism more efficiently.
Results: We applied this approach to a transcriptional regulatory footprint of phthalimide neovascular factor 1 (PNF1)—a novel synthetic small molecule that exhibits significant in vitro endothelial potency—spanning 1–48 h post-supplementation in human micro-vascular endothelial cells (HMVEC) to comprehensively interrogate PNF1 effects. We concluded that PNF1 first induces tumor necrosis factor-alpha (TNF-α) signaling pathway function which in turn affects transforming growth factor-beta (TGF-β) signaling. These results are consistent with our previous observations of PNF1-directed TGF-β signaling at 24 h, including differential regulation of TGF-β-induced matrix metalloproteinase 14 (MMP14/MT1-MMP) which is implicated in angiogenesis. Ultimately, we illustrate how our pathway-based compendium analysis more efficiently generates hypotheses for compound mechanism than existing techniques.
Availability: The microarray data generated as part of this study are available in the Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/).
Contact: email@example.com; firstname.lastname@example.org
Supplementary information: Supplementary data are available at Bioinformatics online.
NGF is a growth factor for which the role in the promotion of angiogenesis is still not completely understood. We found that NGF promotes the pathological neovascularization process in glioma through a direct interaction with α9β1 integrin, which is up-regulated on microvascular endothelial cells in cancer tissue. We propagated gHMVEC primary cells using a new method of immune-selection, and these cells demonstrated α9β1 integrin-dependent binding of NGF in a cell adhesion assay. Moreover, NGF induced gHMVEC proliferation and chemotaxis inhibited by specific blockers of α9β1 integrin, such as MLD-disintegrins and monoclonal antibody Y9A2. A Matrigel tube formation assay revealed that NGF significantly increased capillary-like growth from gHMVEC to a level comparable to treatment with VEGF. The snake venom disintegrin, VLO5, inhibited the agonistic effect of both growth factors, whereas the effect of Y9A2 was not statistically significant. Angiogenesis exogenously induced by NGF was also α9β1-integrin dependent in an embryonic quail CAM system. However, angiogenesis pathologically induced by developing glioma in this system was only sensitive for inhibition with MLD-disintegrin, suggesting a more complex effect of cancer cells on the neovascularization process. The anti-angiogenic effect of MLD-disintegrins is probably related to their pro-apoptotic ability induced in activated tumoral endothelial cells. Therefore, the molecular basis of these disintegrins may be useful for developing new angiostatic pharmaceuticals for application in cancer therapy.
angiogenesis; disintegrins; glioma; integrins; nerve growth factor
During neovascularization, the end result is a new functional microcirculation comprised of a network of mature microvessels with specific topologies. While much is known concerning the mechanisms underlying the initiation of angiogenesis, it remains unclear how the final architecture of microcirculatory beds is regulated. To begin to address this, we determined the impact of angiogenic neovessel pre-patterning on the final microvascular network topology using an implant model of implant neovascularization.
Methods and Results
To test this, we used 3-D direct-write bioprinting or physical constraints in a manner permitting post-angiogenesis vascular remodeling and adaptation to pattern angiogenic microvascular precursors (neovessels formed from isolated microvessel segments) in 3-dimensional collagen gels prior to implantation and subsequent network formation. Neovasculatures pre-patterned into parallel arrays formed functional networks following 4 weeks post-implantation, but lost the pre-patterned architecture. However, maintenance of uniaxial physical constraints during post-angiogenesis remodeling of the implanted neovasculatures produced networks with aligned microvessels as well as an altered proportional distribution of arterioles, capillaries and venules.
Here we show that network topology resulting from implanted microvessel precursors is independent from pre-patterning of precursors but can be influenced by a patterning stimulus involving tissue deformation during post-angiogenesis remodeling and maturation.
microcirculation; regeneration; bioprinting; vascular engineering; neovascularization
Plexiform neurofibromas (PNF) are benign tumors of the peripheral nerve which mostly develop in patients with neurofibromatosis type 1 (NF1). Surgical interventions are usually not applied to children with small tumors. These are rather restricted to debulking of larger tumors in adults that cause clinical complications or aesthetic disfigurement. In most cases, a total resection of PNF is not possible due to the network-like growth of the tumors.
Patients and methods
Early surgical intervention was carried out for 9 small PNFs in 7 NF1 children. Tumor resection was performed following the graphical delineation of the affected skin and according the MRI findings.
Total resection was achieved for all 9 PNF without causing any neurological or organic deficit. Annual magnetic resonance tomography over a period of four years did not reveal any relapse of the tumors.
Early surgical intervention for small superficial PNFs in NF1 children have various advantages and may especially be considered a strategy to prevent progression.
Shoot growth and development is mediated by the activity of the shoot meristem, which initiates leaves and axillary meristems. Meristem maintenance is achieved by a poorly understood process that functions to sustain the balance of stem cell perpetuation in the central zone (CZ) and organogenesis in the peripheral zone (PZ). A recent study showed that two related homeodomain transcription factors, PENNYWISE (PNY) and POUND-FOOLISH (PNF), regulate meristem maintenance by controlling the integrity of the CZ. The non-flower producing phenotype displayed by pny pnf plants can be rescued by genetically increasing the size of the shoot meristem. In this addendum, we show that augmenting the size of the central region of pny pnf shoot meristems partially rescues the meristem termination phenotype that occurs during early stages of vegetative development. Thus, regulation of CZ integrity by PNY and PNF is crucial for vegetative and reproductive development.
development; shoot growth; meristem; homeodomain; stem cells
We have previously demonstrated that implanted microvessels form a new microcirculation with minimal host-derived vessel investment. Our objective was to define the vascular phenotypes present during neovascularization in these implants and identify post-angiogenesis events. Morphological, functional and transcriptional assessments identified three distinct vascular phenotypes in the implants: sprouting angiogenesis, neovascular remodeling, and network maturation. A sprouting angiogenic phenotype appeared first, characterized by high proliferation and low mural cell coverage. This was followed by a neovascular remodeling phenotype characterized by a perfused, poorly organized neovascular network, reduced proliferation, and re-associated mural cells. The last phenotype included a vascular network organized into a stereotypical tree structure containing vessels with normal perivascular cell associations. In addition, proliferation was low and was restricted to the walls of larger microvessels. The transition from angiogenesis to neovascular remodeling coincided with the appearance of blood flow in the implant neovasculature. Analysis of vascular-specific and global gene expression indicates that the intermediate, neovascular remodeling phenotype is transcriptionally distinct from the other two phenotypes. Therefore, this vascular phenotype likely is not simply a transitional phenotype but a distinct vascular phenotype involving unique cellular and vascular processes. Furthermore, this neovascular remodeling phase may be a normal aspect of the general neovascularization process. Given that this phenotype is arguably dysfunctional, many of the microvasculatures present within compromised or diseased tissues may not represent a failure to progress appropriately through a normally occurring neovascularization phenotype.
neovascularization; neovessel; sprouting; angiogenesis; microvascular remodeling; microcirculation; structural adaptation; gene expression
AIM: To investigate the regulation of mindin expression and the signaling pathway involved during inflammation.
METHODS: C57BL/6 mice were treated with 3% dextran sulfate sodium (DSS) in drinking water for 6 d to induce acute colitis, and then the colon was harvested for histological analysis or for RNA isolation. mRNA expression of mindin and nuclear factor (NF)-κB p65 was analyzed by quantitative real time polymerase chain reaction (RT-PCR) and mindin expression construct was confirmed by Western blotting. Mouse macrophage and intestinal epithelial lineage cells were stimulated with different cytokines and toll-like receptor (TLR) ligands, before pNF-κB-luciferase activity was assessed using the Dual-Luciferase reporter assay system.
RESULTS: mRNA expression of mindin was upregulated 4.7 ± 1.1 fold compared with the baseline during DSS-induced intestinal inflammation in the mice. Stimulation with CpG-ODN (a known TLR-9 ligand) induced 4.2 ± 0.3 fold upregulation of mindin expression in RAW 264.7 cells. Full-length of mindin was cloned from cDNA of mouse mesenteric lymph node, then the pCMV-Mindin-Flag expression vector was established and the protein expression level was confirmed. Transfection of the mindin construct and stimulation with CpG-ODN significantly increased the NF-κB-luciferase activity by 2.5 ± 0.3 and 4.5 ± 0.5 fold in RAW264.7 and CMT93 cells, respectively (P < 0.01).
CONCLUSION: Mindin expression is upregulated during intestinal inflammation and may induce NF-κB promoter activation in a TLR-9 mediated manner.
Mindin; Nuclear factor-κB promoter activity; Toll-like receptor-9
Introduction. The proprioceptive neuromuscular facilitation (PNF) is a physiotherapeutic concept based on muscle and joint proprioceptive stimulation. Among its principles, the irradiation is the reaction of the distinct regional muscle contractions to the position of the application of the motions. Objective. To investigate the presence of irradiated dorsiflexion and plantar flexion and the existing strength generated by them during application of PNF trunk motions. Methods. The study was conducted with 30 sedentary and female volunteers, the PNF motions of trunk flexion, and extension with the foot (right and left) positioned in a developed equipment coupled to the load cell, which measured the strength irradiated in Newton. Results. Most of the volunteers irradiated dorsal flexion in the performance of the flexion and plantar flexion during the extension motion, both presenting an average force of 8.942 N and 10.193 N, respectively. Conclusion. The distal irradiation in lower limbs became evident, reinforcing the therapeutic actions to the PNF indirect muscular activation.
Engineered vascularized adipose tissue could serve as an alternative to traditional tissue reconstruction procedures. Adipose formation occurs in a coordinated fashion with neovascularization. Previous studies have shown that extracellular matrix-based materials supplemented with factors that stimulate neovascularization promote adipogenesis in a number of animal models. The present study examines the ability of fibroblast growth factor (FGF-1) delivered from alginate microbeads to induce neovascularization and adipogenesis in type I collagen gels in a vascular pedicle model of adipose tissue engineering. FGF-1 loaded microbeads stimulated greater vascular network formation in an in vitro 3D co-culture model compared than a single bolus of FGF-1. In in vivo studies, FGF-1 loaded beads suspended in collagen and implanted in a chamber surrounding the exposed femoral pedicle of a rat resulted in a significant increase in vascular density at 1 and 6 weeks in comparison to bolus administration of FGF-1. Staining for smooth muscle actin showed that over 48% of vessels had associated mural cells. While an increase in neovascularization was achieved, there was less than 3% adipose under any condition. These results show that delivery of FGF-1 from alginate beads stimulated a more persistent neovascularization response than bolus FGF-1 both in vitro and in vivo. However, unlike previous studies, this increased neovascularization did not result in adipogenesis. Future studies need to provide a better understanding of the relationship between neovascularization and adipogenesis in order to design advanced tissue engineering therapies.
Neovascularization facilitates tumour growth and metastasis formation. In our laboratory, we attempt to identify clinically available oral efficacious drugs for antiangiogenic activity. Here, we report which non-steroidal anti-inflammatory drugs (NSAIDs) can inhibit corneal neovascularization, induced by basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF). This antiangiogenic activity may contribute to the known effects of NSAIDs on gastric ulcers, polyps and tumours. We found that sulindac was one of the most potent antiangiogenic NSAIDs, inhibiting bFGF-induced neovascularization by 50% and VEGF-induced neovascularization by 55%. Previously, we reported that thalidomide inhibited growth factor-induced corneal neovascularization. When we combined sulindac with thalidomide, we found a significantly increased inhibition of bFGF- or VEGF-induced corneal neovascularization (by 63% or 74% respectively) compared with either agent alone (P< 0.01). Because of this strong antiangiogenic effect, we tested the oral combination of thalidomide and sulindac for its ability to inhibit the growth of V2 carcinoma in rabbits. Oral treatment of thalidomide or sulindac alone inhibited tumour growth by 55% and 35% respectively. When given together, the growth of the V2 carcinoma was inhibited by 75%. Our results indicated that oral antiangiogenic combination therapy with thalidomide and sulindac may be a useful non-toxic treatment for cancer. © 1999 Cancer Research Campaign
basic fibroblast growth factor; vascular endothelial growth factor; corneal neovascularization; non-steroidal anti-inflammatory drug; cyclo-oxygenase
Microvascular remodeling is a complex process that includes many cell types and molecular signals. Despite a continued growth in the understanding of signaling pathways involved in the formation and maturation of new blood vessels, approximately half of all compounds entering clinical trials will fail, resulting in the loss of much time, money, and resources. Most pro-angiogenic clinical trials to date have focused on increasing neovascularization via the delivery of a single growth factor or gene. Alternatively, a focus on the concerted regulation of whole networks of genes may lead to greater insight into the underlying physiology since the coordinated response is greater than the sum of its parts. Systems biology offers a comprehensive network view of the processes of angiogenesis and arteriogenesis that might enable the prediction of drug targets and whether or not activation of the targets elicits the desired outcome. Systems biology integrates complex biological data from a variety of experimental sources (-omics) and analyzes how the interactions of the system components can give rise to the function and behavior of that system. This review focuses on how systems biology approaches have been applied to microvascular growth and remodeling, and how network analysis tools can be utilized to aid novel pro-angiogenic drug discovery.
The purpose of this survey was for comparison with a similar 1981 survey to determine if proprioceptive neuromuscular facilitation (PNF) techniques are being implemented in the same manner today.
Design and Setting:
The survey was made available at the 1993 NATA Clinical Symposium.
The subjects were 131 athletic trainers representing all major national athletic conferences who attended the 1993 NATA Clinical Symposium and who stated that they used PNF exercise in their practice.
The survey consisted of 15 questions dealing with academic preparation, years of practice, scope and method of preparation in PNF, application of nine PNF techniques to various joints and regions of the body, and the most successful use of PNF techniques.
PNF techniques are most frequently applied during rehabilitation of the knee, shoulder, and hip, similar to 1981 except that the use of these techniques during ankle rehabilitation has increased. In both studies, the most frequently used techniques were contract-relax and hold-relax. Two techniques not surveyed in 1981, contract-relax-contract and hold-relax-contract, are becoming techniques of choice for elbow, wrist, hip, and knee rehabilitation. The use of PNF techniques in the muscle re-education phase of rehabilitation is an application identified in this survey not cited by athletic trainers in the 1981 survey.
Proprioceptive and kinesthetic deficits are known to occur after certain types of injuries, and the use of PNF techniques to correct these problems is a natural application A contemporary trend in exercise rehabilitation is multiplanar exercises, which are typified by PNF techniques.
proprioception; neuromuscular; facilitation; injuries
sFlt-1 (soluble Flt-1) potently inhibits angiogenesis by binding extracellularly to VEGF (vascular endothelial growth factor). In the present paper, we report that hypoxia down-regulates sFlt-1 expression in HMVECs (human microvascular endothelial cells), a constituent of microvessels where angiogenesis occurs. Hypoxia (5–1% O2) increased VEGF expression in HMVECs. In contrast, the levels of sFlt-1 mRNA and protein in HMVECs decreased significantly as the O2 concentration fell, whereas mFlt-1 (membrane-bound Flt-1) mRNA and protein remained unchanged. This suggested that hypoxia selectively regulates alternative 3′-end processing of sFlt-1 pre-mRNA. We have also demonstrated that sFlt-1 overexpression in lentiviral-construct-infected HMVECs counteracted VEGF-induced endothelial cell growth. We next identified cis-elements involved in sFlt-1 mRNA processing in HMVECs using a human Flt-1 minigene and found that two non-contiguous AUUAAA sequences function as the poly(A) signal. Furthermore, we identified a cis-element in intron 13 that regulates sFlt-1 mRNA processing. Mutagenesis of the U-rich region in intron 13 caused a significant decrease in the soluble-form/membrane-form RNA ratio in the minigene-transfected HMVECs. These results suggest that decreased sFlt-1 expression due to hypoxia contributes to hypoxia-induced angiogenesis and reveals a novel mechanism regulating angiogenesis by alternative mRNA 3′-end processing.
angiogenesis; hypoxia; mRNA 3′-end processing; soluble Flt-1; vascular endothelial cell; vascular endothelial growth factor (VEGF); ARE, A+U-rich element; bFGF, basic fibroblast growth factor; CFIM25, 25-kDa subunit of the cleavage factor Im; CstF, cleavage stimulation factor; Cst F64, 64-kDa subunit of CstF; DMOG, dimethyloxaloylglycine; EC, endothelial cell; FBS, fetal bovine serum; HEK-293TN cell, HEK (human embryonic kidney)-293 cell expressing the large T-antigen of SV40 (simian virus 40) and the neomycin-resistance gene; HIF, hypoxia-inducible factor; hnRNP, heterogeneous nuclear ribonucleoprotein; HUVEC, human umbilical vein EC; KDR, kinase insert domain-containing receptor; mFlt-1, membrane-bound Flt-1; MVEC, microvascular EC; HMVEC, human MVEC; PHD, prolyl hydroxylase; RT, reverse transcription; sFlt-1, soluble Flt-1; VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor; sVEGFR, soluble VEGFR
Therapeutic angiogenesis is a promising strategy to promote the formation of new or collateral vessels for tissue regeneration and repair. Since changes in tissue oxygen concentrations are known to stimulate numerous cell functions, these studies have focused on the oxygen microenvironment and its role on the angiogenic potential of endothelial cells. We analyzed the proangiogenic potential of human endothelial colony-forming cells (hECFCs), a highly proliferative population of circulating endothelial progenitor cells, and compared outcomes to human dermal microvascular cells (HMVECs) under oxygen tensions ranging from 1% to 21% O2, representative of ischemic or healthy tissues and standard culture conditions. Compared to HMVECs, hECFCs (1) exhibited significantly greater proliferation in both ischemic conditions and ambient air; (2) demonstrated increased migration compared to HMVECs when exposed to chemotactic gradients in reduced oxygen; and (3) exhibited comparable or superior proangiogenic potential in reduced oxygen conditions when assessed using a vessel-forming assay. These data demonstrate that the angiogenic potential of both endothelial populations is influenced by the local oxygen microenvironment. However, hECFCs exhibit a robust angiogenic potential in oxygen conditions representative of physiologic, ischemic, or ambient air conditions, and these findings suggest that hECFCs may be a superior cell source for use in cell-based approaches for the neovascularization of ischemic or engineered tissues.
Angiogenesis; Endothelial cells; Endothelial progenitor cells; Neovascularization
Nicotinic acetylcholine receptors (nAChR) are best known for their role in neurotransmission, but they have recently been demonstrated on vascular endothelial cells. Acetylcholine is their endogenous ligand, but they are also stimulated by nicotine. By stimulating nAChR, nicotine promotes tumor angiogenesis as well as atherosclerotic plaque neovascularization. In this study, we investigated the role of nAChR in the pathogenesis of choroidal neovascularization (CNV).
The effect of the nAChR antagonist mecamylamine was tested on human retinal and choroidal endothelial cells in vitro and in a murine model of CNV.
Several nAChR isoforms were identified in retinal and choroidal microvascular endothelial cells and the ability of these cells to form tubules when grown in Matrigel and VEGF was suppressed by the nAChR antagonist mecamylamine. Supplementation of the drinking water of mice with nicotine increased the size of CNV lesions at Bruch’s membrane rupture sites, an effect that was blocked by infusion of mecamylamine by osmotic pump. In the absence of nicotine, CNV formation was suppressed by mecamylamine given by osmotic pump or by topical application to the cornea.
These data suggest that endogenous activation of nAChR promotes CNV and activation of nAChR by nicotine may contribute to the increased incidence of CNV seen in smokers with age-related macular degeneration (AMD). Topically administered mecamylamine could provide an appealing new treatment approach for CNV.
Angiogenesis, or neovascularization, is known to play an important role in the neoplastic progression leading to metastasis. CD31 or Factor VIII-related antigen (F VIII RAg) immunohistochemistry (IHC), is widely used in experimental studies quantifing tumor neovascularization in immunocompromised animal models implanted with transformed human cell lines. Quantification, however, can be affected extensively by variations in the methodology used to measure vascularization including antibody selection, pretreatment with antigen retrieval and evaluation techniques. To examine this further, we examined the microvessel density and the intensity of microvascular staining among five different human tumor xenografts and a mouse syngeneic tumor using anti-CD31 and F VIII RAg IHC staining. Different antigen retrieval methods also were evaluated. Maximal retrieval of CD31 was achieved using 0.5 M Tris (pH 10) buffer, while maximum retrieval of F VIII RAg was achieved using 0.05% pepsin treatment of tissue sections. For each optimized retrieval condition, compared to F VIII RAg, anti-CD31 highlighted small vessels better. Furthermore, the microvessel density of CD31 was significantly greater than that of F VIII RAg decorated vessels (p < 0.001). The choice of antibody and antigen retrieval method has a significant affect on immunohistochemical findings when studying angiogenesis. One also must use caution when comparing studies in the literature that use different techniques and reagents.
angiogenesis; antigen retrieval; CD31/PECAM-1; endothelial cells; factor VIII/vWf; immunohistochemistry; microvessel density; xenografts
Antiangiogenic therapy for the treatment of cancer and other neovascular diseases is desired to be selective for pathological angiogenesis and lymphangiogenesis. Macrophage colony-stimulating factor (M-CSF), a cytokine required for the differentiation of monocyte lineage cells, promotes the formation of high-density vessel networks in tumors and therefore possesses therapeutic potential as an M-CSF inhibitor. However, the physiological role of M-CSF in vascular and lymphatic development, as well as the precise mechanisms underlying the antiangiogenic effects of M-CSF inhibition, remains unclear. Moreover, therapeutic potential of M-CSF inhibition in other neovascular diseases has not yet been evaluated. We used osteopetrotic (op/op) mice to demonstrate that M-CSF deficiency reduces the abundance of LYVE-1+ and LYVE1− macrophages, resulting in defects in vascular and lymphatic development. In ischemic retinopathy, M-CSF was required for pathological neovascularization but was not required for the recovery of normal vasculature. In mouse osteosarcoma, M-CSF inhibition effectively suppressed tumor angiogenesis and lymphangiogenesis, and it disorganized extracellular matrices. In contrast to VEGF blockade, interruption of M-CSF inhibition did not promote rapid vascular regrowth. Continuous M-CSF inhibition did not affect healthy vascular and lymphatic systems outside tumors. These results suggest that M-CSF–targeted therapy is an ideal strategy for treating ocular neovascular diseases and cancer.
Pigment epithelium-derived factor (PEDF) was first identified in retinal pigment epithelium cells. It is an endogenously produced protein that is widely expressed throughout the human body such as in the eyes, liver, heart, and adipose tissue; it exhibits multiple and varied biological activities. PEDF is a multifunctional protein with antiangiogenic, antitumorigenic, antioxidant, anti-inflammatory, antithrombotic, neurotrophic, and neuroprotective properties. More recently, PEDF has been shown to be the most potent inhibitor of stem/progenitor cell-associated neovascularization. Neovascularization is a complex process regulated by a large, interacting network of molecules from stem/progenitor cells. PEDF is also involved in the pathogenesis of angiogenic eye disease, tumor growth, and cardiovascular disease. Novel antiangiogenic agents with tolerable side effects are desired for the treatment of patients with various diseases. Here, we review the value of PEDF as an important endogenous antiangiogenic molecule; we focus on the recently identified role of PEDF as a possible new target molecule to influence stem/progenitor cell-related neovascularization.
TNFα is a proinflammatory cytokine that plays a central role in the pathogenesis of rheumatoid arthritis (RA). We investigated the effects of certolizumab pegol, a TNFα blocker, on endothelial cell function and angiogenesis.
Human dermal microvascular endothelial cells (HMVECs) were stimulated with TNFα with or without certolizumab pegol. TNFα-induced adhesion molecule expression and angiogenic chemokine secretion were measured by cell surface ELISA and angiogenic chemokine ELISA, respectively. We also examined the effect of certolizumab pegol on TNFα-induced myeloid human promyelocytic leukemia (HL-60) cell adhesion to HMVECs, as well as blood vessels in RA synovial tissue using the Stamper-Woodruff assay. Lastly, we performed HMVEC chemotaxis, and tube formation.
Certolizumab pegol significantly blocked TNFα-induced HMVEC cell surface angiogenic E-selectin, vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 expression and angiogenic chemokine secretion (P < 0.05). We found that certolizumab pegol significantly inhibited TNFα-induced HL-60 cell adhesion to HMVECs (P < 0.05), and blocked HL-60 cell adhesion to RA synovial tissue vasculature (P < 0.05). TNFα also enhanced HMVEC chemotaxis compared with the negative control group (P < 0.05) and this chemotactic response was significantly reduced by certolizumab pegol (P < 0.05). Certolizumab pegol inhibited TNFα-induced HMVEC tube formation on Matrigel (P < 0.05).
Our data support the hypothesis that certolizumab pegol inhibits TNFα-dependent leukocyte adhesion and angiogenesis, probably via inhibition of angiogenic adhesion molecule expression and angiogenic chemokine secretion.
Corneal neovascularization is a serious condition that can lead to a profound decline in vision. The abnormal vessels block light, cause corneal scarring, compromise visual acuity, and may lead to inflammation and edema. Corneal neovascularization occurs when the balance between angiogenic and antiangiogenic factors is tipped toward angiogenic molecules. Vascular endothelial growth factor (VEGF), one of the most important mediators of angiogenesis, is upregulated during neovascularization. In fact, anti-VEGF agents have efficacy in the treatment of neovascular age-related macular degeneration, diabetic retinopathy, macular edema, neovascular glaucoma, and other neovascular diseases. These same agents have great potential for the treatment of corneal neovascularization. We review some of the most promising anti-VEGF therapies, including bevacizumab, VEGF trap, siRNA, and tyrosine kinase inhibitors.
anti-VEGF therapy; corneal neovascularization; bevacizumab; ranibizumab; VEGF Trap; silencing RNA
Previous studies indicate that green tea extract may inhibit breast cancer progression by blocking angiogenesis, although the molecular mechanisms are not well defined. We demonstrate that administration of Polyphenon E (Poly E), a standardized green tea extract, inhibited MDA-MB231 breast cancer and human dermal microvascular endothelial (HMVEC) cell migration and the expression of vascular endothelial growth factor (VEGF) and matrix metalloproteinase 9 (MMP9). In addition, Poly E inhibited VEGF-induced neovascularization in vivo. We also demonstrate that Poly E blocked signal transducers and activators of transcription (STAT) signaling by suppressing interferon-gamma (IFN-γ)-induced gene transcription via IFN-γ-activating sequence (GAS) elements and downstream STAT3 activation by inhibiting STAT1 and STAT3 dimerization in MDA-MB231 cells. Transient expression of constitutively active STAT3 significantly reduced the inhibitory effect of Poly E on cell migration and VEGF and MMP9 expression. Taken together, these observations indicate that green tea extract inhibits angiogenesis partly through the disruption of STAT3-mediated transcription of genes, including VEGF.
Green tea; Breast cancer; STAT3; Angiogenesis
Transcription factors (TFs) and miRNAs are the most important dynamic regulators in the control of gene expression in multicellular organisms. These regulatory elements play crucial roles in development, cell cycling and cell signaling, and they have also been associated with many diseases. The Regulatory Network Analysis Tool (RENATO) web server makes the exploration of regulatory networks easy, enabling a better understanding of functional modularity and network integrity under specific perturbations. RENATO is suitable for the analysis of the result of expression profiling experiments. The program analyses lists of genes and search for the regulators compatible with its activation or deactivation. Tests of single enrichment or gene set enrichment allow the selection of the subset of TFs or miRNAs significantly involved in the regulation of the query genes. RENATO also offers an interactive advanced graphical interface that allows exploring the regulatory network found.RENATO is available at: http://renato.bioinfo.cipf.es/.
Angiogenesis and microcirculation play a central role in growth and metastasis of human neoplasms, and, thus, represent a major target for novel treatment strategies. Mechanistic analysis of processes involved in tumor vascularization, however, requires sophisticated in vivo experimental models and techniques. Intravital microscopy allows direct assessment of tumor angiogenesis, microcirculation and overall perfusion. Its application to the study of tumor-induced neovascularization further provides information on molecular transport and delivery, intra- and extravascular cell-to-cell and cell-to-matrix interaction, as well as tumor oxygenation and metabolism. With the recent advances in the field of bioluminescence and fluorescent reporter genes, appropriate for in vivo imaging, the intravital fluorescent microscopic approach has to be considered a powerful tool to study microvascular, cellular and molecular mechanisms of tumor growth.
cranial window; dorsal skinfold chamber; in vivo; perfusion; vascularization