Voluntary running suppresses proinflammatory cytokines and bone marrow endothelial progenitor cell levels in apolipoprotein-E-deficient mice

Long-term exercise is associated with reduced atherosclerotic burden, inflammation, and enhanced endothelial progenitor cell (EPC) levels in mice. Infusion of progenitor cells in mice decreases atherosclerosis and suppresses inflammation. The aim of this study was to determine whether exercise-induced enhancement of EPCs is associated with reduced atherosclerosis and inflammation. To study this, 20-week old ApoE(-/-) mice with advanced atherosclerotic lesions (n = 12/group) were randomized to voluntary running or no running for 8 weeks. Exercise led to a potent suppression of elevated circulating proinflammatory cytokines without significant reduction of atherosclerotic lesions. When repeated in ApoE(-/-) mice with early atherosclerotic disease, exercise led to a 62% (p = 0.017) reduction in lesion thickness (intima-to-media ratio) at the aortic root. Interestingly, BM-EPC levels were significantly elevated under proinflammatory conditions seen in ApoE(-/-) mice and decreased in response to exercise, independent of the degree of atherosclerosis. Under early atherosclerotic conditions, long-term exercise reduces atherosclerotic plaque burden and is associated with reduced systemic inflammation. Elevated BM-EPCs seen in atherosclerotic conditions may be a marker of generalized vascular inflammation or injury, and decrease in response to exercise, along with other markers of inflammation.     

Galectin-3 gene inactivation reduces atherosclerotic lesions and adventitial inflammation in ApoE-deficient mice

This study has examined the role of galectin-3 (GaL3), a multicompartmented N-acetyllactosamine-binding chimeric lectin, on atherogenesis in the ApoE-deficient mouse model of atherosclerosis. Pathological changes consisting of atheromatous plaques, atherosclerotic microaneurysms extending into periaortic vascular channels, and adventitial and periaortic inflammatory infiltrates were assessed in an equal number (n = 36) of apolipoprotein (Apo)E-deficient mice and ApoE-GaL3 double-knockout mice. These mice were divided into three age groups, 21 to 23 weeks, 25 to 31 weeks, and 36 to 44 weeks of age. Results of this morphological analysis have shown an age-related increase in the incidence of aorta atheromatous plaques and periaortic vascular channels in ApoE-deficient mice. By contrast ApoE/GaL3 double-knockout mice did not show an increase in pathological changes with age. The 36- to 44-week group of ApoE(-/-)/GaL3(-/-) mice had a significantly lower number of atherosclerotic lesions (P < 0.004) and fewer atheromatous plaques (P < 0.008) when compared with ApoE(-/-)/GaL3+/+ mice of the same age. ApoE(-/-)/GaL3(-/-) mice had a lower number of perivascular inflammatory infiltrates and mast cells than those found in ApoE(-/-)/GaL3+/+ mice. The reduced number of perivascular mast cells may have resulted in a low level of interleukin-4 that contributed to the reduction in the morphological parameters of atherogenesis correlated with the lack of GaL3 expression. The effect of GaL3 deficiency on atherogenesis decrease could be related to its function as a multifunctional protein implicated in macrophage chemotaxis, angiogenesis, lipid loading, and inflammation.     

Oxidatively damaged DNA in aging dyslipidemic ApoE-/- and wild-type mice

The free radical theory of aging depicts an accumulation of cellular oxidatively damaged DNA. In this study, we investigated this theory in mice with knocked-out apolipoprotein E gene (ApoE(-/-)), which develops atherosclerosis and wild-type counterparts. The level of oxidatively damaged DNA was investigated as strand breaks, endonuclease III- and formamidopyrimidine DNA glycosylase-sensitive sites by the comet assay. The level of DNA damage was mainly increased with age in the liver of ApoE(-/-) mice, whereas no increase was observed in the aorta or lung of the mice. This suggests that the accumulation of oxidized DNA in the liver of dyslipidemic ApoE(-/-) mice could be secondary to dysfunction of the lipid metabolism. Visually, the aortas of the ApoE(-/-) mice were clearly atherosclerotic as indicated by rigid texture and yellowish in color. However, the unaltered levels of oxidized DNA in severely atherosclerotic aortas of old ( approximately 70 weeks) ApoE(-/-) mice indicate that oxidative stress may not be a generalized phenomenon, but rather related locally to the individual plaques. In conclusion, the results of this study suggest that dyslipidemic ApoE(-/-) mice suffer from hepatic oxidative stress in terms of oxidized DNA, and this effect could be due to the dysfunction of lipid metabolism.     

Genetic loss of Gas6 induces plaque stability in experimental atherosclerosis

The growth arrest-specific gene 6 (Gas6) plays a role in pro-atherogenic processes such as endothelial and leukocyte activation, smooth muscle cell migration and thrombosis, but its role in atherosclerosis remains uninvestigated. Here, we report that Gas6 is expressed in all stages of human and mouse atherosclerosis, in plaque endothelial cells, smooth muscle cells and macrophages. Gas6 expression is most abundant in lesions containing high amounts of macrophages, ie thin fibrous cap atheroma and ruptured plaque. Genetic loss of Gas6 does not affect the number and size of initial and advanced plaques in ApoE(-/-) mice, but alters its plaque composition. Compared to Gas6(+/+): ApoE(-/-) mice, initial and advanced plaques of Gas6(-/-): ApoE(-/-) mice contained more smooth muscle cells and more collagen and developed smaller lipid cores, while the expression of TGFbeta was increased. In addition, fewer macrophages were found in advanced plaques of Gas6(-/-): ApoE(-/-) mice. Hence, loss of Gas6 promotes the formation of more stable atherosclerotic lesions by increasing plaque fibrosis and by attenuating plaque inflammation. These findings identify a role for Gas6 in plaque composition and stability.     

Dietary restriction reduces atherosclerosis and oxidative stress in the aorta of apolipoprotein E-deficient mice

Dietary restriction (DR) has been shown to inhibit almost all the age-related diseases, e.g. cardiomyopathy and cancers, in rodents. However, there is little information for the effect of DR on atherosclerosis. In the present study, we examined the effect of DR on the development of atherosclerosis in mice homozygous knockout for apolipoprotein E gene (ApoE(-/-)). The ApoE(-/-) mice were fed either ad libitum (AL) or 60% of the diet consumed by the mice fed AL. Atherosclerotic lesions in the proximal aorta of these mice were measured. Our results showed that ApoE(-/-) mice fed the calorie-restricted diet had smaller and relatively early stages of atherosclerotic lesions (e.g. foam cells and free lipids) when compared to ApoE(-/-) mice fed AL, who developed more advanced lesions (e.g. fibrous caps and acellular areas). In addition, ApoE(-/-) mice fed the calorie-restricted diet showed a significant decrease in the level of lipid hydroperoxides and the production of superoxide and hydrogen peroxide in the aorta as compared to ApoE(-/-) mice fed AL. These observations suggest that reduction of oxidative stress in the arterial wall may contribute to the anti-atherogenic effect of DR in ApoE(-/-) mice.     

Requirement of apelin-apelin receptor system for oxidative stress-linked atherosclerosis

The recently identified endogenous peptide apelin and its specific apelin receptor (APJ) are currently being considered as potential regulators in vascular tissue. Previously, we reported apelin mediates phosphorylation of myosin light chain and elicits vasoconstriction in vascular smooth muscle. In this study, physiological roles of the apelin-APJ system were investigated on atherosclerosis. In APJ and apolipoprotein E double-knockout (APJ(-/-)ApoE(-/-)) mice fed a high-cholesterol diet, atherosclerotic lesions were dramatically reduced when compared with APJ(+/+) ApoE(-/-) mice, in the absence of an effect of cholesterol levels. Immunohistochemical detection of smooth muscle cells, using a smooth muscle alpha-actin antibody, showed greatly reduced staining for these cells in lesions of APJ(-/-)ApoE(-/-) mice fed a high-cholesterol diet. Vascular production of superoxide radicals and the expression of nicotinamide-adenine dinucleotide phosphate oxidase subunits were decreased in APJ(-/-)ApoE(-/-) mice compared with APJ(+/+)ApoE(-/-) mice fed a standard normal diet. In vascular smooth muscle cells, apelin induced nicotinamide-adenine dinucleotide phosphate oxidase subunit expression. Apelin also induced vascular smooth muscle cell proliferation, which was inhibited by superoxide dismutase or diphenylene iodonium. The apelin-APJ system is a mediator of oxidative stress in vascular tissue, and thus we propose it to be a critical factor in atherogenesis under high-cholesterol dietary conditions. APJ deficiency is preventative against oxidative stress-linked atherosclerosis.     

Age-related intimal stiffening enhances endothelial permeability and leukocyte transmigration

Age is the most significant risk factor for atherosclerosis; however, the link between age and atherosclerosis is poorly understood. During both aging and atherosclerosis progression, the blood vessel wall stiffens owing to alterations in the extracellular matrix. Using in vitro and ex vivo models of vessel wall stiffness and aging, we show that stiffening of extracellular matrix within the intima promotes endothelial cell permeability--a hallmark of atherogenesis. When cultured on hydrogels fabricated to match the elasticity of young and aging intima, endothelial monolayers exhibit increased permeability and disrupted cell-cell junctions on stiffer matrices. In parallel experiments, we showed a corresponding increase in cell-cell junction width with age in ex vivo aortas from young (10 weeks) and old (21 to 25 months) healthy mice. To investigate the mechanism by which matrix stiffening alters monolayer integrity, we found that cell contractility increases with increased matrix stiffness, mechanically destabilizing cell-cell junctions. This increase in endothelial permeability results in increased leukocyte extravasation, which is a critical step in atherosclerotic plaque formation. Mild inhibition of Rho-dependent cell contractility using Y-27632, an inhibitor of Rho-associated kinase, or small interfering RNA restored monolayer integrity in vitro and in vivo. Our results suggest that extracellular matrix stiffening alone, which occurs during aging, can lead to endothelial monolayer disruption and atherosclerosis pathogenesis. Because previous therapeutics designed to decrease vascular stiffness have been met with limited success, our findings could be the basis for the design of therapeutics that target the Rho-dependent cellular contractile response to matrix stiffening, rather than stiffness itself, to more effectively prevent atherosclerosis progression.     

Angiogenic and antiangiogenic balance regulates concomitant antitumoral resistance

Concomitant antitumoral resistance (CAR), the phenomenon by which the growth of distant secondary tumor implants or metastases in some tumor-bearing hosts is inhibited by the presence of a primary tumor, has been previously ascribed to an antiangiogenic process. Here, we investigated vascular endothelial growth factor (VEGF) and endostatin serum levels in nude or BALB/c mice bearing human lung tumors (Calu-6 and H460) or murine mammary tumors (M3MC, M-234p and M-234m), respectively. In these experimental models we previously found an association between in vivo generation of CAR and in vitro conversion of plasminogen into angiostatin. Serum endostatin level in CAR+ Calu-6-bearing mice was significantly higher than in CAR- H460 counterpart. Sera from mammary tumor-bearing mice showed similar levels of endostatin, regardless of their ability to induce CAR. Conversely, serum VEGF levels in mice bearing CAR+ tumors were lower than those found in CAR- tumor-bearing hosts. Immunostaining with an anti-CD31 antibody revealed that secondary tumors subjected to CAR were significantly less vascularized than primary tumors, while this difference was not observed in CAR- tumors. In vitro studies showed an inhibitory effect of sera from CAR-inducing tumors on endothelial cell proliferation as compared to normal sera, whereas sera from non-CAR-inducing tumors did not alter endothelial proliferation and, in some instances, even caused stimulation of endothelial proliferation. These data suggest that the antiangiogenic mechanism operating in concomitant antitumoral resistance is the result of an increase in the ratio of antiangiogenic/proangiogenic regulators. The levels of the factors involved in this phenomenon can vary in the different tumor models, but the trend favoring the inhibition of angiogenesis is always conserved.     

An assay for macrophage-mediated regulation of endothelial cell proliferation

We have developed an assay that quantifies the potential of macrophages to regulate proliferation of endothelial cells. We show that young mice macrophages can be distinguished from old mice macrophages by their ability to inhibit vascular endothelial cell proliferation. While young mice macrophages robustly inhibit proliferation, old mice macrophages fail to do so and actually promote the proliferation of endothelial cells. In this report, we outline a technique that directly assesses the effect of macrophages on modulation of endothelial cell proliferation. This assay will help us in understanding the mechanisms of macrophage function in several disease states characterized by abnormal angiogenesis including cancers, angiogenic eye disease and atherosclerotic heart disease.     

Activation of poly(ADP-ribose) polymerase contributes to the endothelial dysfunction associated with hypertension and aging

Increased production of reactive oxygen and nitrogen species has recently been implicated in the pathogenesis of endothelial dysfunction associated with atherosclerosis, hypertension and aging. Oxidant induced cell injury triggers the activation of nuclear enzyme poly(ADP-ribose) polymerase (PARP), which in turn contributes to cardiac and vascular dysfunction in various pathophysiological conditions including diabetes, reperfusion injury and circulatory shock. Here we investigated the role of PARP activation in the pathogenesis of cardiac and endothelial dysfunction associated with atherosclerosis, hypertension and aging. Retired breeder spontaneously hypertensive rats (SHR, 40 weeks old) and apolipoprotein E knockout mice (apoE-Ko, 10 weeks old) were treated for 20 weeks with vehicle or the potent PARP inhibitor PJ34. In the vehicle-treated SHR rats and apoE-Ko mice (kept on atherogenic diet) there was a significant loss of endothelial function, as measured by the relaxant responsiveness of vascular rings to acetylcholine. SHR rats also developed severe hypertension and cardiac hypertrophy. Treatment with the PARP inhibitor did not influence high blood pressure and cardiac hypertrophy in SHR rats, but it improved Ach-induced, NO-mediated vascular relaxation. In addition to the beneficial effects of chronic treatment with PARP inhibitor, 1-h in vitro incubation of aortic rings from SHR rats with PJ34 (3 micromol/l) was also able to improve the endothelial dysfunction. In contrast, in apoE-Ko mice PJ34 treatment did not affect the parameters studied. Thus, PARP activation contributes to the pathogenesis of endothelial dysfunction associated with hypertension and aging, but not in the current experimental model of atherosclerosis.     

Efficacy of anti-angiogenic treatment of tumors in old versus young mice

Cancer treatment in the older population, the most afflicted by the disease, is as yet, inefficient. A reduced aggressiveness of tumors is often observed in the elderly, implying the necessity for therapeutic modalities adjusted to age. A rational design of age-related cancer therapy could be based on the mechanisms of this phenomenon. It is suggested that, in addition to the patient's old age-specific health problems (which prohibit the use of the aggressive cancer treatments now in use), the age-related differential tumor biology (apparently beneficial to the old) should also be considered for the design of treatment modalities suitable for the aged. Based on one mechanism of the reduced aggressiveness of tumors in the old (age-dependent decreased angiogenesis), we compared the effect of an anti-angiogenic treatment in young and old mice. TNP-470 treatment resulted in an inhibitory effect on B16 melanoma in both young and old mice but the effect was more pronounced in old animals. Moreover, a high percentage of long-term surviving animals was observed only in the old-treated mice. Treatment with TNP-470 of the AKR lymphoma produced similar results. We thus found a differential age-dependent therapeutic efficiency of an anti-angiogenic agent on two tumors. Importantly, the anti-angiogenic drug was more efficient against tumors of old animals.     

The usefulness of mouse breast tumor models for testing and optimization of breast cancer vaccines at old age

Cancer is an age-related disease and with the graying of the society, there is an increasing need to optimize cancer management and therapy for application in elderly patients. Cancer vaccines that can be applied in both prevention and therapy are potentially less toxic than chemotherapy or radiation and could, therefore, be especially suitable for older more frail cancer patients. In this study, we used syngeneic metastatic (4TO7) and non-metastatic (64pT) breast tumor models to obtain valuable information on the potential usefulness of MAGE-encoding cancer vaccines in metastatic and non-metastatic breast cancer at old age. First, we tested a mouse Mage-b DNA vaccine in young mice and found a significant preventive effect on the development of metastases. However, little effect was observed on primary breast tumors. Second, we studied tumor progression in relation to aging and found significant smaller tumors in old compared to young mice. This was associated with an increase in the percentage of CD8(+) T cells in the inguinal lymph nodes at the site of the tumor at old age. These findings suggest that breast cancer immunotherapeutic approaches could be a valid strategy even in elderly patients.     

Defects in activation of nitric oxide synthases occur during delayed angiogenesis in aging

Angiogenesis, the formation of new vessels from pre-existing vasculature, is impaired in aging. This is due, in part, to a lack of regulatory molecules such as nitric oxide (NO). We wished to test the hypothesis that there are deficits in the pathways that mediate NO production during angiogenesis (as defined by fibrovascular invasion into a polyvinyl alcohol (PVA) sponge implant), in aged mice in comparison to young mice. Sponges were implanted subcutaneously in young (6-8 months old, n=11) and aged (23-25 months old, n=13) mice and sampled at 14 and 19 days. Sections from the implants were stained with antibodies against vascular endothelial growth factor receptor 2 (VEGFR-2), Akt, phosphorylated Akt (p-Akt), endothelial nitric oxide synthase (eNOS), phosphorylated eNOS (p-eNOS), inducible NOS (iNOS), and 3-nitrotyrosine (3-NT, a marker for nitrosylated proteins). Expression of VEGFR-2 was similar in the sponges of young and aged mice. Moreover, there were no significant differences in levels of Akt or its phosphorylated form in sponges from young and aged mice at 14 and 19 d. In marked contrast, levels of eNOS, p-eNOS and iNOS were significantly decreased in sponges from aged mice relative to young mice (p<0.02 for eNOS, p-eNOS and <0.01 for iNOS between young and aged mice). Concomitantly, there was diminished expression of 3-NT in the sponges from aged mice (p<0.05). Our data indicate that defects in the activation of nitric oxide synthases result in decreased NO production in aged tissues relative to young tissues. We propose that the subsequent lack of NO contributes to impaired angiogenesis in aging.     

Impaired ATP-induced coronary blood flow and diminished aortic NTPDase activity precede lesion formation in apolipoprotein E-deficient mice

Intravascular ATP and ADP are important regulators of vascular tone, thrombosis, inflammation, and angiogenesis. This study was undertaken to evaluate the contribution of purinergic signaling to disturbed vasodilation and vascular remodeling during atherosclerosis progression. We used apolipoprotein E-deficient (Apoe(-/-)) mice as an appropriate experimental model for atherosclerosis. Noninvasive transthoracic Doppler echocardiography imaging with adenosine, ATP, and other nucleotides and nonhydrolyzable P2 receptor agonists and antagonists suggests that ATP regulates coronary blood flow in mice through activation of P2Y (most likely, endothelial ATP/UTP-selective P2Y(2)) receptors, rather than via its dephosphorylation to adenosine. Strikingly, compared to age-matched wild-type controls, young (10- to 15-week-old) Apoe(-/-) mice displayed diminished coronary reactivity in response to ATP but not adenosine. The impaired hyperemic response to ATP persisted in older (20- to 30-week-old) Apoe(-/-) mice, which were additionally characterized by mild atherosclerosis (as ascertained by aortic Oil Red O staining) and a systemic increase in plasma ATP and ADP levels. Concurrent thin-layer chromatographic analysis of nucleoside triphosphate diphosphohydrolase (NTPDase) and ecto-5'-nucleotidase/CD73 activities in thoracic aortas, lymph nodes, spleen, and serum revealed that aortic NTPDase was decreased by 40% to 50% in a tissue-specific manner both in young and mature Apoe(-/-) mice. Collectively, disordered purinergic signaling in Apoe(-/-) mice may serve as important prerequisite for impaired blood flow, local accumulation of ATP and ADP at sites of atherogenesis, and eventually, the exacerbation of atherosclerosis.     

Reduced acute vascular injury and atherosclerosis in hyperlipidemic mice transgenic for lysozyme

Hyperlipidemia promotes oxidant stress, inflammation, and atherogenesis in apolipoprotein E-deficient (ApoE((-/-))) mice. Mice transgenic for lysozyme (LZ-Tg) are resistant to acute and chronic oxidative stress and have decreased circulating levels of pro-oxidant advanced glycation end-products (AGEs). Herein we report that TIB-186 macrophages transduced with adenovirus-expressing human LZ (AdV-LZ) containing the AGE-binding domain facilitated AGE uptake and degradation and that AdV-LZ-transduced macrophages and peritoneal macrophages from LZ-Tg mice suppressed the AGE-triggered tumor necrosis factor-alpha response. We assessed atherosclerosis in LZ-Tg mice crossed with ApoE((-/-)) mice (LZ/ApoE((-/-))) and found increased serum LZ levels and decreased AGE and 8-isoprostanes levels, although hyperlipidemia remained similar to ApoE((-/-)) controls. Atherosclerotic plaques and neointimal lesions at the aortic root and descending aorta were markedly decreased (by 40% and 80%, respectively) in LZ/ApoE((-/-)) versus ApoE((-/-)) mice, as were inflammatory infiltrates. The arterial lesions following femoral artery injury in LZ/ApoE((-/-)) mice were suppressed (intimal to media ratio decreased by 50%), as were AGE deposits and vascular smooth muscle cell activation, compared to ApoE((-/-)) mice. Despite hyperlipidemia, development of atheroma and occlusive, inflammatory arterial neointimal lesions in response to injury was suppressed in LZ/ApoE((-/-)) mice. This effect may be due to the antioxidant properties of LZ, which is possibly linked to the AGE-binding domain region of the molecule.     

T-kininogen induces endothelial cell proliferation

Basal proliferation of endothelial cells increases with age, and this might play a role in the etiology of age-related vascular diseases, as well as angiogenesis. Serum kininogen levels increase during aging in rats and humans, and T-kininogen (T-KG) can affect proliferative homeostasis in several cell models. Both kinins and kininogens have been shown previously to be angiogenic through activation of endothelial cell proliferation, and here we show that exposure of endothelial cells to T-KG results in vigorous cell proliferation, accompanied by ERK/AKT activation. In our experiments, the proliferative response requires B1 and B2 kinin receptors, even though kinins are not released from the precursor. We hypothesize that the age-related increase in T-KG could play a significant role in the age-related dysregulation of vascular physiology and function.     

Granulocyte colony-stimulating factor promotes tumor angiogenesis via increasing circulating endothelial progenitor cells and Gr1+CD11b+ cells in cancer animal models

Recombinant granulocyte colony-stimulating factor (G-CSF) is used for cancer patients with myelosuppression induced by chemotherapy. G-CSF has been reported to progress tumor growth and angiogenesis, but the precise mechanism of tumor angiogenesis activated by G-CSF has not been fully clarified. N-terminal-mutated recombinant human G-CSF administration increased WBCs and neutrophils in peripheral blood and reduced bone marrow stromal cell-derived factor-1 in mice, indicating its biological relevance. Mice were inoculated with Lewis lung carcinoma cells (LLCs) or KLN205 cells and treated with G-CSF. G-CSF accelerated tumor growth and intratumoral vessel density, while it did not accelerate proliferation of LLCs, KLN205 cells or human umbilical vein endothelial cells in vitro. In the absence of tumors, G-CSF did not increase circulating cells that displayed phenotypic characteristics of endothelial progenitor cells (EPCs). In the presence of tumors, G-CSF increased circulating EPCs. In addition, G-CSF treatment increased immune suppressor and endothelial cell-differentiating Gr1+CD11b+ cells in tumor-bearing mice. We conclude that G-CSF promotes tumor growth by activating tumor angiogenesis via increasing circulating EPCs and Gr1+CD11b+ cells in cancer animal models.     

Reduced atherosclerotic lesion size in P-selectin deficient apolipoprotein E-knockout mice fed a chow but not a fat diet

P-selectin. We investigated the role of P-selectin on the development of vascular lesions in an ApoE(-/-) male mice. Double-knockout (ApoE(-/-), P-selectin(-/-); DKO) were compared to single-knockout (ApoE(-/-); SKO) mice. They were fed a chow or fat diet for 3, 6, 15, and 20 weeks, without any differences in cholesterol levels. DKO mice fed a chow diet exhibited a ratio of lesion area over media lower than SKO mice, for 3 (P < .03) , 6 (P < .001), and 15 (P < .02) weeks. DKO mice fed a fat diet showed a lower ratio only at 3 weeks. P-selectin deficiency in ApoE(-/-) mice has a protective effect in atherosclerotic lesions development. Reduction of lesion size depends on diet type and duration. A fat diet could neutralize the beneficial effects of P-selectin deficiency, inducing atherosclerotic lesions via probably other adhesion molecules.     

N-acetyl-cysteine promotes angiostatin production and vascular collapse in an orthotopic model of breast cancer

The antioxidant N-acetyl-cysteine (NAC) has been shown to be chemopreventive in clinical studies, and in recent studies, has shown promise in preventing tumor progression. Although the effects of NAC on tumorigenesis have been associated with decreased angiogenesis, the mechanism of the anti-angiogenic activity has not been determined. In the following study, we describe a novel mechanism whereby NAC therapy blocks MDA-MB-435 breast carcinoma cell proliferation and metastasis in an in vivo tumorigenic model. Athymic nude mice bearing MDA-MB-435 xenografts were treated with systemic NAC daily for 8 weeks. NAC treatment resulted in endothelial cell apoptosis and reduction of microvascular density within the core of the tumor leading to significant tumor cell apoptosis/necrosis. Angiostatin accumulated in tumors from NAC-treated but not control animals. Additional studies using a vascular endothelial growth factor-dependent chicken chorioallantoic membrane angiogenic assay recapitulated NAC-induced endothelial apoptosis and coordinate production of angiostatin, a potent endothelial apoptotic factor. In vitro studies showed angiostatin was formed in endothelial cultures in a vascular endothelial growth factor- and NAC-dependent manner, a process that requires endothelial cell surface plasminogen activation. These results suggest that systemic NAC therapy promotes anti-angiogenesis through angiostatin production, resulting in endothelial apoptosis and vascular collapse in the tumor.