Negative Angiogenic Factors Research Articles

Effects of detraining on the temporal expression of positive and negative angioregulatory proteins in skeletal muscle of mice.

Temporal expression of positive and negative angiogenic factors in response to detraining is poorly understood. We report the protein expression of anti-angiogenic peptides (thrombospondin-1, TSP-1; and endostatin) as well as pro-angiogenic factors (vascular endothelial growth factor, VEGF; matrix metalloproteinases-2 and -9), and nucleolin (a nuclear protein involved with synthesis and maturation of ribosomes) in response to detraining in triceps surae muscles of C57BL/6 mice. Male mice were allowed to exercise voluntarily for 21days, and then basal and acute response to exercise were evaluated at 1, 7, 14 and 28days detraining (D1, D7, D14, D28, respectively, n=12/group). As seen in the D1 mice, training resulted in the increased muscle capillary-to-fibre ratio (C/F), increased maximal running time and elevated basal expression of VEGF and matrix metalloproteinase-9 (P<0.05). After 7days of detraining (D7), C/F levels were similar to control levels, but both basal VEGF and TSP-1 were elevated (P<0.05). At D14 and D28, TSP-1 protein was not different compared to baseline levels; however, VEGF was elevated in gastrocnemius (GA), but not the soleus (SOL) or plantaris (PLT) muscles, of D14 mice. Endostatin tended to decrease in D14 and D28 compared to controls. Timing of nucleolin protein expression differed between muscle groups, with increases at D1, D7 and D14 in the PLT, SOL and GA muscles, respectively. The response of VEGF and nucleolin to acute exercise was blunted with training, and remained blunted in the PLT and SOL even after 28days of detraining, at a time point long after muscle capillarization was observed to be similar to pre-training levels. These data suggest that TSP-1 may be a mediator of capillary regression with detraining, even in the face of elevated VEGF, suggesting that pro-angiogenic regulators may not be able to prevent the regression of skeletal muscle capillaries under physiological conditions. The responses of matrix metalloproteinases, endostatin and nucleolin poorly correlated with detraining-induced capillary regression.

Temporal response of positive and negative regulators in response to acute and chronic exercise training in mice

Angiogenesis is controlled by a balance between positive and negative angiogenic factors, but temporal protein expression of many key angiogenic regulators in response to exercise are still poorly defined. In C57BL/6 mice, we evaluated the temporal protein expression of several pro-angiogenic and anti-angiogenic factors in response to (1) a single acute bout of exercise and (2) chronic exercise training resulting from 3, 5, 7, 14 and 28 days of voluntary wheel running. Following acute exercise, protein levels of vascular endothelial growth factor-A (VEGF), endostatin and nucleolin were increased at 2-4 h (P < 0.05), whereas matrix metalloproteinase (MMP)-2 was elevated within a 12-24 h window (P < 0.05). Training increased muscle capillarity 11%, 15% and 22% starting with 7, 14 and 28 days of training, respectively (P < 0.01). Basal VEGF and MMP-2 were increased by 31% and 22%, respectively, compared to controls (P < 0.05) after 7 days (7d) training, but decreased to back to baseline after 14d training. After 28d training VEGF fell 49% below baseline control (P < 0.01). Basal muscle expression of thrombospondin 1 (TSP-1) was ∼900% greater in 14d- and 28d-trained mice compared to either 5d- and 7d-trained mice (P < 0.05), and tended to increase by ∼180-258% compared to basal control levels (P < 0.10). The acute responsiveness of VEGF to exercise in untrained mice (i.e. 161% increase, P < 0.001) was lost with capillary adaptation occurring after 7, 14 and 28d training. Taken together, these data support the notion that skeletal muscle angiogenesis is controlled by a balance between positive and negative mitogens, and reveals a complex, highly-coordinated, temporal scheme whereby these factors can differentially influence capillary growth in response to acute versus chronic exercise.

Basal expression of VEGF does not correlate with loss of skeletal muscle capillarity with detraining

Temporal expression of positive and negative angiogenic factors in response to training and detraining is still poorly described. Protein expression of anti‐angiogenic thrombospondin‐1 (TSP‐1) and endostatin as well as pro‐angiogenic vascular endothelial growth factor (VEGF), matrix metalloproteinases ‐2, ‐9 (MMP‐2, MMP‐ 9), and nucleolin (a nuclear protein involved with synthesis and maturation of ribosomes) were examined after training and detraining in mouse gastrocnemius muscle. Mice were placed in one of 5 groups (n=12/group): control (did not run), trained (voluntary wheel ran 21 days), or detrained (21 day trained, then detrained) for 7, 14, or 28 days. When compared to control mice, training increased capillary‐to‐fiber ratio (C:F) and resulted in an increase of basal VEGF and MMP‐9 (p&lt;0.05), all other factors remained unchanged. After 7 days of detraining, C:F returned to control levels, basal VEGF remained elevated and TSP‐1 increased (p&lt;0.05). Following 14 days of detraining, basal VEGF levels were still elevated, TSP‐1 fell to baseline, and nucleolin declined compared to controls (p&lt;0.05). After 28 days of detraining, basal VEGF returned to baseline, TSP‐1 remained unchanged while endostatin and MMP‐2 levels decreased compared to controls (p&lt;0.05). These data suggest that an increase in TSP‐1 and a decrease in MMP's and nucleolin, despite elevated VEGF, correlate with detraining losses of skeletal muscle capillarity. Supported by NIH 5P20RR016477; NIH 5T32HL090610; AHA 10BGIA3630002

Importance of Anti‐angiogenic Factors in the Regulation of Skeletal Muscle Angiogenesis

The microcirculation is essential for delivery of oxygen and nutrients to maintain skeletal muscle health and function. The network of microvessels surrounding skeletal myocytes has a remarkable plasticity that ensures a good match between muscle perfusion capacities and myofiber metabolic needs. Depending on physiologic conditions, this vascular plasticity can either involve growth (e.g., exercise-induced angiogenesis) or regression (e.g., physical deconditioning) of capillaries. This angio-adaptative response is thought to be controlled by a balance between pro- and anti-angiogenic factors and their receptors. While changes in the expression or activity for pro-angiogenic factors have been well studied in response to acute and chronic exercise during the past two decades, little attention thus far has been devoted to endogenous negative regulators that are also likely to be important in regulating capillary growth/regression. Indeed, the importance and contribution of anti-angiogenic factors in controlling skeletal muscle angiogenesis remains poorly understood. Here, we highlight the emerging research related to skeletal muscle expression of several negative angiogenic factors and discuss their potential importance in controlling skeletal muscle angio-adaptation, particularly in physiologic response to physical activity.

Angiogenesis and Lymphangiogenesis of Gastric Cancer

Tumor angiogenesis is the result of an imbalance between positive and negative angiogenic factors released by tumor and host cells into the microenvironment of the neoplastic tissue. The stroma constitutes a large part of most solid tumors, and cancer-stromal cell interactions contribute functionally to tumor growth and metastasis. Activated fibroblasts and macrophages in tumor stroma play important roles in angiogenesis and tumor progression. In gastric cancer, tumor cells and stromal cells produce various angiogenic factors, including vascular endothelial growth factor, interleukin-8, platelet-derived endothelial cell growth factor, and angiopoietin. In addition, Helicobacter pylori infection increases tumor cell expression of metastasis-related genes including those encoding several angiogenic factors. We review the current understanding of molecular mechanisms involved in angiogenesis and lymphangiogenesis of human gastric cancer.

Cancer-Stromal Cell Interaction and Tumor Angiogenesis in Gastric Cancer

Recent studies in molecular and cellular biology have shown that tumor growth and metastasis are not determined by cancer cells alone but also by a variety of stromal cells. The stroma constitutes a large part of most solid tumors, and cancer-stromal cell interaction contributes functionally to tumor growth and metastasis. Angiogenesis is the result of an imbalance between positive and negative angiogenic factors released by tumor and host cells into the microenvironment of the neoplastic tissue. In gastric cancer, tumor cells and stromal cells produce various angiogenic factors, including vascular endothelial growth factor, interleukin-8, and platelet-derived endothelial cell growth factor. The microenvironment in the gastric mucosa may also influence the angiogenic phenotype of gastric cancer. Helicobacter pylori infection increases expression of several angiogenic factors by tumor cells. Activated fibroblasts and macrophages in tumor stroma also play an important role in angiogenesis and tumor progression. We review the current understanding of cancer-stromal cell interaction as it pertains to tumor angiogenesis in gastric cancer.

Prognostic Value of Angiogenic Factors (Vacular Endothelial Growth Factor [VEGF] and Basic Fibroblast Growth Factor [bFGF]) and Endostatin in Patients with Non-Hodgkin Lymphoma.

Introduction: Tumor angiogenesis is gaining importance in hematological malignancies; it is balanced by many known and unknown positive and negative angiogenic factors. However, studies related to the prognostic significance of angiogenic factors and lymphoproliferative diseases are limited compared to solid tumors. This is a single institution study evaluating the prognostic impact of serum Endostatin, VEGF, and bFGF in non-Hodgkin's lymphoma (NHL).Patients and methods: Pretreatment serum level of Endostatin (S-Endostatin), VEGF (S-VEGF), and bFGF (S-bFGF) were measured by Enzyme-Linked Immunoabsorbant Assay (ELISA) on 136 NHL patients: 82 patients (60%) had aggressive NHL (diffuse large B cell lymphoma [DLBCL] or other aggressive non-DLBCL) and 54 patients (40%) had indolent lymphomas (follicular lymphoma [FL] or other indolent non-FL). For each angiogenic factor, univariate progression-free survival (PFS) and overall survival (OS) analyses were performed according to the quartiles of the distribution of serum level values. Each of the analyses was aimed to detect a trend between prognosis and the serum level of the angiogenic factor. Eight prognostic groups were defined according to the type of NHL and the prognostic indexes (International Prognostic Index [IPI] for DLBCL and aggressive non-DLBCL, and Follicular International Prognostic Index [FLIPI] for FL). Survival analyses stratified on the prognostic group were used to test the independent prognostic value of each angiogenic factor. Survival curves were compared with logrank tests. Distributions of serum level values were compared with Kruskal-Wallis tests. The median follow-up was 78 months.Results: Pretreatment angiogenic serum levels were not significantly different between all categories of NHL, except for VEGF level, which was slightly higher in the aggressive group compared to the indolent group (666 pg/ml vs. 465 pg/ml; p=0.03). Low S-Endostatin and high S-VEGF at diagnosis were associated with poor PFS (p=0.002 and p=10−4 respectively) and poor OS (p=0.04 and p=10−6 respectively). Patients with S-Endostatin within the lowest quartile had only 29% PFS and 49% OS in contrast to a 67% PFS and 82% OS among patients with Endostatin within the highest quartile. Similarly, patients with S-VEGF within the highest quartile had only 20% PFS and 26% OS in contrast to a 70% PFS and 85% OS among patients with VEGF within the lowest quartile. The VEGF/Endostatin ratio was used to combine the results from S-VEGF and S-Endostatin. The 4 groups corresponding to the quartiles of the distribution of VEGF/Endostatin ratio had different OS and PFS (p<10−6 for both); PFS and OS were respectively 15% and 16% for patients from the highest quartile VEGF/Endostatin ratio and 79% and 84% for those from the lowest quartile Figure 1. The independent prognostic impact of S-Endostatin, S-VEGF and VEGF/Endostatin ratio remained significant on PFS (p=0.0003, p=0.001 and p<10−6 respectively) and OS (p=0.01, p=10−4 and p<10−6 respectively) after stratification on the prognostic group. We did not find any relation between pretreatment S-bFGF and prognosis in our patients.Conclusion: Our results showed that pretreatment serum levels of VEGF and Endostatin are significantly related to outcome in NHL patients; the higher the VEGF/Endostatin ratio is, the poorer the prognosis. A better understanding of angiogenesis in lymphoproliferative diseases is mandatory in order to develop new anti-angiogenic targeted therapeutic agents that can change the outcome of these patients. [Display omitted]

Microvessel density and regulators of angiogenesis in malignant and nonmalignant prostate tissue

The aim of this study was to investigate the relationship between microvessel density (MVD), positive and negative angiogenic factors, and established prognostic factors in prostate cancer (PC), and, to clarify the effect of angiogenic factors to angiogenesis. The vascularization of neoplastic, non-neoplastic prostate tissue was determined by CD34 immunostaining. Angiogenetic mediators VEGF, bFGF, TSP-1, and p53 were studied by immunohistochemistry. Neovascularization and p53, VEGF, and TSP-1 expressions of tumorous tissue were higher than non-tumorous tissue. The bFGF expression in these tissues was not different. The p53 expression was not correlated with the expressions of VEGF, bFGF, and TSP-1 in PC. Our results demonstrate a significant increase in MVD, VEGF, TSP-1, and p53 expressions in prostate tumorigenesis. The pretreatment sPSA was the only parameter demonstrating significant correlation with tumor grade and may have a value in the prediction of aggressive tumor behavior in PC.

Circulating Level of Vascular Endothelial Growth Factor in Differentiating Hemangioma from Vascular Malformation Patients

The majority of vascular anomalies can be diagnosed accurately based on natural history and physical examination; however, there is no convenient, noninvasive, and objective method to (1) differentiate hemangioma from vascular malformation; (2) determine whether a hemangioma is in the proliferating or involuting phase; (3) tell whether or not corticosteroids or interferon alfa-2a is effective for hemangioma; or (4) follow up hemangioma. Although the differences in endothelial cell, protein, and mRNA expression levels of some positive and negative angiogenic factors in the lesions can help to solve these problems, these methods (pathological section, immunohistochemical analysis, and in situ hybridization techniques) necessitate that a biopsy be performed, and the procedures are complicated. A nonsurgical and convenient method would have significant clinical applications. Fifty-nine patients with proliferating hemangiomas, 38 with involuting hemangiomas, 18 with vascular malformations, and 12 negative control subjects were examined for serum levels of vascular endothelial growth factor using enzyme-linked immunosorbent assays. The serum level of vascular endothelial growth factor in proliferating hemangiomas was significantly higher than that in involuting hemangiomas, vascular malformations, and negative controls, while differences among involuting hemangiomas, vascular malformations, and negative controls were not statistically significant. In addition, after systemic steroid therapy, the serum level of vascular endothelial growth factor was significantly reduced compared with pretreatment levels in six patients with proliferating hemangiomas. The serum level of vascular endothelial growth factor may be useful in differentiating hemangioma from vascular malformations, staging hemangiomas, judging the efficacy of steroid therapy, and evaluating follow-up criteria for hemangiomas. The results probably shed new light on the pathogenesis of hemangiomas.

Modulation of the angiogenesis response through Ha-ras control, placenta growth factor, and angiopoietin expression in mouse skin carcinogenesis.

Tumor angiogenesis is governed by a complex balance of positive and negative angiogenic factors. Development of chemically-induced mouse skin tumors appears to be highly dependent on an early burst of neovascularization. We have previously shown that Ha-ras-driven vascular endothelial growth factor (VEGF) expression plays a pivotal role in this process. However, the status of other critical positive and negative angiogenic factors throughout skin tumorigenesis has not been studied to the same extent. In the present study, we show that another VEGF family member, placenta growth factor (PlGF), was highly upregulated at all tumor stages in a ras-dependent manner. The study of angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2), ligands of receptor tyrosine kinase 2 (Tie-2), showed that while stroma-derived Ang-2 was increased, epidermal Ang-1 expression was completely abolished at early papilloma formation. Studies using epidermal tumor cell lines suggest that the disappearance of Ang-1 also depends on ras activation, extending the plethora of events controlled by this oncogene in mouse skin carcinogenesis. Our results indicated that tumor development occurred in a strong angiogenesis-prone scenario in which PlGF and Ang-2 acted cooperatively with VEGF, whereas the negative or stabilizing effect of Ang-1 was abrogated. A time-course sequence of expression of angiogenic factors expressed throughout tumor growth, as well as the identification of key signaling molecules triggering the angiogenic response, may contribute to the development and testing of antiangiogenic therapeutic strategies with this in vivo tumor model.

Tissue factor expression and angiogenesisin human prostate carcinoma

In tumors, the switch to the angiogenic phenotype is thought to be controlled by a balance of positive and negative angiogenic factors. Tissue factor (TF) produced by tumor cells has been implicated in the regulation of this “angiogenic switch” through its ability to concurrently induce the expression of angiogenic molecules such as vascular endothelial cell growth factor (VEGF), while inhibiting the expression of anti-angiogenic molecules such as thrombospondin 2. We have examined TF expression and its relationship to angiogenesis and tumor progression in human prostate carcinomas. Most of the prostate carcinoma specimens examined (73%; n = 67) express high levels of TF Immunohistochemical analysis localized TF expression to the epithelial cells of malignant glands. TF expression was significantly correlated with tumor angiogenesis as measured by the microvessel density (MVD). In addition, TF expression was correlated with the preoperative PSA level, a strong predictor of recurrence in prostate carcinomas. Our findings show that TF expression by the malignant glands in prostate cancer is common and suggest a role for this molecule in regulating prostate cancer progression and angiogenesis.

Assignment of Transforming Growth Factor β1 and β3 and a Third New Ligand to the Type I Receptor ALK-1

Germ line mutations in one of two distinct genes, endoglin or ALK-1, cause hereditary hemorrhagic telangiectasia (HHT), an autosomal dominant disorder of localized angiodysplasia. Both genes encode endothelial cell receptors for the transforming growth factor beta (TGF-beta) ligand superfamily. Endoglin has homology to the type III receptor, betaglycan, although its exact role in TGF-beta signaling is unclear. Activin receptor-like kinase 1 (ALK-1) has homology to the type I receptor family, but its ligand and corresponding type II receptor are unknown. In order to identify the ligand and type II receptor for ALK-1 and to investigate the role of endoglin in ALK-1 signaling, we devised a chimeric receptor signaling assay by exchanging the kinase domain of ALK-1 with either the TGF-beta type I receptor or the activin type IB receptor, both of which can activate an inducible PAI-1 promoter. We show that TGF-beta1 and TGF-beta3, as well as a third unknown ligand present in serum, can activate chimeric ALK-1. HHT-associated missense mutations in the ALK-1 extracellular domain abrogate signaling. The ALK-1/ligand interaction is mediated by the type II TGF-beta receptor for TGF-beta and most likely through the activin type II or type IIB receptors for the serum ligand. Endoglin is a bifunctional receptor partner since it can bind to ALK-1 as well as to type I TGF-beta receptor. These data suggest that HHT pathogenesis involves disruption of a complex network of positive and negative angiogenic factors, involving TGF-beta, a new unknown ligand, and their corresponding receptors.

Progressive growth of infantile cutaneous hemangiomas is directly correlated with hyperplasia and angiogenesis of adjacent epidermis and inversely correlated with expression of the endogenous angiogenesis inhibitor, IFN-beta.

Cutaneous infantile hemangioma progresses through proliferation and involution phases. Since treatment with interferon, a negative regulator of angiogenesis, accelerates the involution phase, we hypothesized that cutaneous infantile hemangioma is associated with an imbalance between endogenous positive and negative regulators of angiogenesis. We examined 30 specimens of cutaneous hemangioma [proliferative phase (n=15), involuting phase (n=8), and involuted phase (n=7)] and control human skin (n=17), fixed in formalin and embedded in paraffin. Routine histology, immunohistochemistry, and an mRNA in situ hybridization technique were used to measure expression of the positive angiogenic molecules basic fibroblast growth factor (bFGF) and vascular endothelial growth factor/vascular permeability factor (VEGF/VPF), and an endogenous inhibitor of angiogenesis, interferon-beta (IFN-beta). Proliferative phase hemangiomas expressed high levels of bFGF and VEGF/VPF but not IFN-beta (mRNA and protein). The epidermis directly overlying proliferating hemangiomas was hyperplastic, contained numerous dividing cells, and expressed bFGF and VEGF/VPF but not IFN-beta. Epidermis from normal individuals and epidermis directly overlying involuted tumors or at sites distant to the proliferating hemangioma was not hyperplastic and expressed normal levels of bFGF, VEGF/VPF, and IFN-beta. These data suggest that the proliferation of cutaneous hemangiomas and adjacent epidermis is associated with an imbalance between positive and negative angiogenic factors expressed by the neoplasm and adjacent normal tissue.

Clinical significance of angiogenic factors in breast cancer.

Growth, progression, and metastasis of breast cancer, as well as of most of the other tumors, are angiogenesis-dependent processes. Several pro-angiogenic growth factors and endogenous inhibitors of angiogenesis have been identified and sequenced, and experimental studies suggest that angiogenic activity of a tumor may result from downregulation of inhibitors of angiogenesis or up-regulation of endothelial growth factors. The mechanisms leading to the alteration of the balance between positive and negative modulators of angiogenesis are only partially known. We are at the beginning of research to identify the more active angiogenic factors in human breast cancer, and little information is presently available on their clinical significance. Preliminary results suggest that among the known angiogenic peptides, both vascular endothelial growth factor (VEGF) and platelet-derived endothelial cell growth factor / thymidine phosphorylase (PD-ECGF/TP) have promising prognostic and, perhaps, predictive value. No data are available on the clinical value of co-determination of positive and negative regulators of angiogenesis to look at the angiogenic balance of each single tumor. Only a few studies have assessed the role of endogenous inhibitors of angiogenesis in human breast cancer, with results available only on thrombospondin-1 and -2 (TSP-1, -2). Finally, the determination of some integrins such as alpha6 and alphavbeta3 and of some other endothelial-adhesion molecules seems to be of potential prognostic value. Recognizing which are the more biologically active positive and negative angiogenic factors is the key for the identification not only of new prognostic markers but also of targets for antiangiogenic therapy in human breast cancer.

Ocular Neovascularization: Clarifying Complex Interactions

Neovascularization within the eye contributes to visual loss in several ocular diseases, the most common of which are proliferative diabetic retinopathy, neovascular age-related macular degeneration, and retinopathy of prematurity. Together, these three diseases afflict persons in all stages of life from birth through late adulthood and account for most instances of legal blindness. Retinopathy of prematurity (ROP) occurs in premature neonates. Normally, the retina becomes completely vascularized at full term. In the premature baby, the retina remains incompletely vascularized at the time of birth. Rather than continuing in a normal fashion, vasculogenesis in the premature neonatal retina becomes disrupted. Abnormal new proliferating vessels develop at the juncture of vascularized and avascular retina. These abnormal new vessels grow from the retina into the vitreous, resulting in hemorrhage and tractional detachment of the retina. Although laser ablation of avascular peripheral retina may halt the neovascular process if delivered in a timely and sufficient manner, some premature babies nevertheless go on to develop retinal detachment. Surgical methods for treating ROP-related retinal detachments in neonates have limited success at this time because of unique problems associated with this surgery, such as the small size of the eyes and the extremely firm vitreoretinal attachments in neonates. Diabetic retinopathy is the leading cause of blindness in adults of working age. In persons with diabetes mellitus, retinal capillary occlusions develop, creating areas of ischemic retina. Retinal ischemia serves as a stimulus for neovascular proliferations that originate from pre-existing retinal venules at the optic disk or elsewhere in the retina posterior to the equator. Severe visual loss in proliferative diabetic retinopathy (PDR) results from vitreous hemorrhage and tractional retinal detachment. Again, laser treatment (panretinal photocoagulation to ischemic retina) may arrest the progression of neovascular proliferations in this disease but only if delivered in a timely and sufficiently intense manner. Some diabetic patients, either from lack of ophthalmic care or despite adequate laser treatment, go on to sustain severe visual loss secondary to PDR. Vitrectomy surgery can reduce but not eliminate severe visual loss in this disease. Age-related macular degeneration is the leading cause of severe visual loss in persons over 65 years old. In contrast to ROP and PDR, in which neovascularization emanates from the retinal vasculature and extends into the vitreous cavity, AMD is associated with neovascularization originating from the choroidal vasculature and extending into the subretinal space. Choroidal neovascularization causes severe visual loss in AMD patients because it occurs in the macula, the area of retina responsible for central vision. The stimuli which lead to choroidal neovascularization are not understood. Laser ablation of the choroidal neovascularization may stabilize vision in selected patients. However, only 10% to 15% of patients with neovascular AMD have lesions judged to be appropriate for laser photocoagulation according to current criteria. Retinopathy of prematurity, proliferative diabetic retinopathy, and neovascular age-related macular degeneration are but three of the ocular diseases which can produce visual loss secondary to neovascularization. Others include sickle cell retinopathy, retinal vein occlusion, and certain inflammatory diseases of the eye. These, however, account for a much smaller proportion of visual loss caused by ocular neovascularization. Additional treatments beyond laser photocoagulation and vitrectomy surgery are needed to improve outcomes in these patients. Pharmacological antiangiogenic therapy can potentially assist in prevention of the onset or progression of ocular neovascularization and is a current goal of many research laboratories and pharmaceutical companies.