Suppression of p53 activity and p21WAF1/CIP1 expression by vascular cell integrin alphaVbeta3 during angiogenesis.

Induction of p53 activity in cells undergoing DNA synthesis represents a molecular conflict that can lead to apoptosis. During angiogenesis, proliferative endothelial cells become apoptotic in response to antagonists of integrin alphavbeta3 and this leads to the regression of angiogenic blood vessels, thereby blocking the growth of various human tumors. Evidence is presented that administration of alphavbeta3 antagonists during angiogenesis in vivo selectively caused activation of endothelial cell p53 and increased expression of the p53-inducible cell cycle inhibitor p21WAF1/CIP1. In vitro studies revealed that the ligation state of human endothelial cell alphavbeta3 directly influenced p53 activity and the bax cell death pathway. Specifically, agonists of endothelial cell alphavbeta3, but not other integrins, suppressed p53 activity, blocked p21WAF1/CIP1 expression, and increased the bcl-2/bax ratio, thereby promoting cell survival. Thus, ligation of vascular cell integrin alphavbeta3 promotes a critical and specific adhesion-dependent cell survival signal during angiogenesis leading to inhibition of p53 activity, decreased expression of p21WAF1/CIP1, and suppression of the bax cell death pathway.     

Molecular profiling of angiogenesis with targeted ultrasound imaging: early assessment of antiangiogenic therapy effects

Molecular ultrasound is capable of elucidating the expression of angiogenic markers in vivo. However, the capability of the method for volumetric "multitarget quantification" and for the assessment of antiangiogenic therapy response has rather been investigated. Therefore, we generated cyanoacrylate microbubbles linked to vascular endothelial growth factor receptor 2 (VEGFR2) and alphavbeta3 integrin binding ligands and quantified their accumulation in squamous cell carcinoma xenografts (HaCaT-ras-A-5RT3) in mice with the quantitative volumetric ultrasound scanning technique, sensitive particle acoustic quantification. Specificity of VEGFR2 and alphavbeta3 integrin binding microbubbles was shown, and changes in marker expression during matrix metalloproteinase inhibitor treatment were investigated. In tumors, accumulation of targeted microbubbles was significantly higher compared with nonspecific ones and could be inhibited competitively by addition of the free ligand in excess. Also, multimarker imaging could successfully be done during the same imaging session. Molecular ultrasound further indicated a significant increase of VEGFR2 and alphavbeta3 integrin expression during tumor growth and a considerable decrease in both marker densities after matrix metalloproteinase inhibitor treatment. Histologic data suggested that the increasing VEGFR2 and alphavbeta3 integrin concentrations in tumors during growth are related to an up-regulation of its expression by the endothelial cells, whereas its decrease under therapy is more related to the decreasing relative vessel density. In conclusion, targeted ultrasound appears feasible for the longitudinal molecular profiling of tumor angiogenesis and for the sensitive assessment of therapy effects in vivo.     

Direct targeting of alphavbeta3 integrin on tumor cells with a monoclonal antibody, Abegrin

The humanized monoclonal antibody Abegrin, currently in phase II trials for treatment of solid tumors, specifically recognizes the integrin alphavbeta3. Due to its high expression on mature osteoclasts, angiogenic endothelial cells, and tumor cells, integrin alphavbeta3 functions in several pathologic processes important to tumor growth and metastasis. Targeting of this integrin with Abegrin results in antitumor, antiangiogenic, and antiosteolytic activities. Here, we exploit the species specificity of Abegrin to evaluate the effects of direct targeting of tumor cells (independent of targeting of endothelia or osteoclasts). Flow cytometry analysis of human tumor cell lines shows high levels of alphavbeta3 on many solid tumors, including cancers of the prostate, skin, ovary, kidney, lung, and breast. We also show that tumor growth of alphavbeta3-expressing tumor cells is inhibited by Abegrin in a dose-dependent manner. We present a novel finding that high-dose administration can actively impair the antitumor activity of Abegrin. We also provide evidence that antibody-dependent cellular cytotoxicity contributes to in vitro and in vivo antitumor activity. Finally, it was observed that peak biological activity of Abegrin arises at serum levels that are consistent with those achieved in clinical trials. These results support a concept that Abegrin can be used to achieve selective targeting of the many tumor cells that express alphavbeta3 integrin. In combination with the well-established concept that alphavbeta3 plays a key role in cancer-associated angiogenesis and osteolytic activities, this triad of activity could provide new opportunities for therapeutic targeting of cancer.     

Targeting ECM-integrin interaction with liposome-encapsulated small interfering RNAs inhibits the growth of human prostate cancer in a bone xenograft imaging model.

The intricate intracellular communication between stromal and epithelial cells, which involves cell-cell-, cell-insoluble extracellular matrix- (ECM), and cell-soluble factor-mediated signaling processes, is an attractive target for therapeutic intervention in hormone-refractory and bone-metastatic prostate cancer. In the present study we demonstrated that androgen-independent PC3 prostate cancer cells adhered to and migrated on vitronectin (VN), a major noncollagenous ECM in mature bone, through the expression of alphav-containing integrin receptors alphavbeta1 and alphavbeta5 on the cell surface, as determined by antibody function blocking assay and flow cytometry analysis. Small interfering RNAs (siRNAs) targeting human integrin alphav markedly reduced their respective mRNA and protein expression in cells, resulting in nearly complete reduction in VN-mediated cancer progression in vitro. In vivo quantitative bioluminescence analysis of human prostate cancer bone xenografts demonstrated for the first time that intratumoral administration of liposome-encapsulated human alphav-siRNAs significantly inhibits the growth of luciferase-tagged PC3 tumors in skeleton, which was associated with decreased integrin alphav expression and increased apoptosis in tumor cells. This integrin-based gene therapy is particularly suitable for the treatment of prostate cancer bone metastasis.     

Angiopoietin-2 differentially regulates angiogenesis through TIE2 and integrin signaling

Angiopoietin-2 (ANG-2) is a key regulator of angiogenesis that exerts context-dependent effects on ECs. ANG-2 binds the endothelial-specific receptor tyrosine kinase 2 (TIE2) and acts as a negative regulator of ANG-1/TIE2 signaling during angiogenesis, thereby controlling the responsiveness of ECs to exogenous cytokines. Recent data from tumors indicate that under certain conditions ANG-2 can also promote angiogenesis. However, the molecular mechanisms of dual ANG-2 functions are poorly understood. Here, we identify a model for the opposing roles of ANG-2 in angiogenesis. We found that angiogenesis-activated endothelium harbored a subpopulation of TIE2-negative ECs (TIE2lo). TIE2 expression was downregulated in angiogenic ECs, which abundantly expressed several integrins. ANG-2 bound to these integrins in TIE2lo ECs, subsequently inducing, in a TIE2-independent manner, phosphorylation of the integrin adaptor protein FAK, resulting in RAC1 activation, migration, and sprouting angiogenesis. Correspondingly, in vivo ANG-2 blockade interfered with integrin signaling and inhibited FAK phosphorylation and sprouting angiogenesis of TIE2lo ECs. These data establish a contextual model whereby differential TIE2 and integrin expression, binding, and activation control the role of ANG-2 in angiogenesis. The results of this study have immediate translational implications for the therapeutic exploitation of angiopoietin signaling.     

Inhibition of endothelial cell survival and angiogenesis by protein kinase A

Receptors for the provisional ECM are important regulators of angiogenesis. One of these receptors, integrin alpha5beta1, plays a critical role in tumor- and growth factor-induced angiogenesis, because antagonists of this integrin potently inhibit angiogenesis and tumor growth. Here we show that the integrin alpha5beta1 promotes endothelial cell survival during angiogenesis in vivo by suppressing the activity of protein kinase A (PKA). Antagonists of integrin alpha5beta1 activate PKA, which then leads to the activation of caspase-8 and induction of apoptosis. Direct activation of PKA by cAMP or by expression of the PKA catalytic subunit also induces endothelial cell apoptosis, resulting in angiogenesis inhibition in vivo. Our studies indicate that ligation of integrin alpha5beta1 during angiogenesis suppresses an apoptotic program that is dependent on PKA. These studies also indicate that induction of endothelial cell apoptosis in vivo by genetic or pharmacological activation of PKA may be a useful strategy to inhibit angiogenesis.     

The future of therapeutic myocardial angiogenesis

Despite improvements in its medical and surgical management, ischemic coronary disease remains responsible for significant morbidity, mortality, and economic burden in developed nations. Therapeutic myocardial angiogenesis is an attractive treatment option for patients with end-stage coronary disease who have failed percutaneous and surgical methods of revascularization. Over the past decade, our understanding of the biology of new blood vessel formation has improved significantly, and consequently, the use of growth factors to induce myocardial angiogenesis has been attempted in preclinical and clinical trials. Although growth factor therapy had demonstrated tremendous success in animal models, clinical trials have shown limited benefit in patients with coronary disease. Vascular endothelial growth factors and fibroblast growth factors are perhaps the most potent inducers of angiogenesis that have been used in animal models, and the only ones that have been used in clinical trials. This review outlines the biology of new vessel formation and the effects of these growth factors in the context of myocardial angiogenesis with an emphasis on the effects on the endothelium. It also provides a brief overview of delivery strategies and summarizes the preclinical and clinical evidence relating to exogenous growth factor delivery for myocardial angiogenesis. Lastly, we discuss the limitations and future challenges of angiogenic therapy.     

RGDN peptide interaction with endothelial alpha5beta1 integrin causes sustained endothelin-dependent vasoconstriction of rat skeletal muscle arterioles.

The ability of an integrin-binding Arg-Gly-Asp-Asn (RGDN)- containing peptide to influence vascular tone by interacting with the alpha5beta1 integrin was studied using rat skeletal muscle arterioles. After blockade of beta3 integrin function, isolated arterioles with spontaneous tone showed concentration-dependent vasoconstrictions to topical application of GRGDNP, a peptide that shows a greater ability to interact with alpha5beta1 than with alphavbeta3. The constriction to GRGDNP (2.1 mM) was inhibited by blocking alpha5 integrin function, and was intensified by blocking beta3 integrin function. In contrast, GRGDSP, a peptide that interacts better with alphavbeta3, was unable to induce sustained constrictions. Removal of the endothelium abolished the vasoconstriction in response to GRGDNP, suggesting that the response was due to release of an endothelium-dependent factor. Indeed, blockade of ETA endothelin receptors with BQ-610 (1 microM), similar to removal of the endothelium and alpha5 integrin blockade, inhibited the vasoconstriction. These data indicate that interaction of RGD peptides, and in particular the RGDN sequence with endothelial cell alpha5beta1, causes endothelin-mediated arteriolar vasoconstriction. These results indicate that integrins are novel signaling receptors within the vascular wall that affect vasomotor tone, and may play an important role in vascular control.     

In vivo imaging shows abnormal function of vascular endothelial growth factor-induced vasculature

Although the angiogenic effect of vascular endothelial growth factor (VEGF) is widely recognized, a central question concerns whether the vessels formed on its overexpression effectively increase tissue perfusion in vivo. To explore this issue, here we exploit AAV vectors to obtain the prolonged expression of VEGF and angiopoietin-1 (Ang1) in rat skeletal muscle. Over a period of 6 months, muscle blood flow (MBF) and vascular permeability were measured by positron emission tomography and single-photon emission computed tomography, respectively. All measurements were performed under resting conditions and after electrically induced muscle exercise. Despite the potent angiogenic effect of VEGF, documented by vessel counting and intravascular volume assessment, the expression of this factor did not improve resting MBF, and it even decreased perfusion after exercise. This deleterious effect was related to the formation of leaky vascular lacunae, which accounted for the occurrence of arteriovenous shunts that excluded the downstream microcirculation. These effects were significantly counteracted by the coinjection of VEGF and Ang1, which determined a marked increase in resting MBF and, most notably, a significant improvement after exercise that persisted over time. Taken together, these results challenge the effectiveness of VEGF as a sole factor to induce angiogenesis and suggest the use of factor combinations to achieve competent vessel formation.     

Sema3E-PlexinD1 signaling selectively suppresses disoriented angiogenesis in ischemic retinopathy in mice

During development, the retinal vasculature grows toward hypoxic areas in an organized fashion. By contrast, in ischemic retinopathies, new blood vessels grow out of the retinal surfaces without ameliorating retinal hypoxia. Restoration of proper angiogenic directionality would be of great benefit to reoxygenize the ischemic retina and resolve disease pathogenesis. Here, we show that binding of the semaphorin 3E (Sema3E) ligand to the transmembrane PlexinD1 receptor initiates a signaling pathway that normalizes angiogenic directionality in both developing retinas and ischemic retinopathy. In developing mouse retinas, inhibition of VEGF signaling resulted in downregulation of endothelial PlexinD1 expression, suggesting that astrocyte-derived VEGF normally promotes PlexinD1 expression in growing blood vessels. Neuron-derived Sema3E signaled to PlexinD1 and activated the small GTPase RhoJ in ECs, thereby counteracting VEGF-induced filopodia projections and defining the retinal vascular pathfinding. In a mouse model of ischemic retinopathy, enhanced expression of PlexinD1 and RhoJ in extraretinal vessels prevented VEGF-induced disoriented projections of the endothelial filopodia. Remarkably, intravitreal administration of Sema3E protein selectively suppressed extraretinal vascular outgrowth without affecting the desired regeneration of the retinal vasculature. Our study suggests a new paradigm for vascular regeneration therapy that guides angiogenesis precisely toward the ischemic retina.     

Angiogenic therapy for coronary artery and peripheral arterial disease

Neovascularization in chronically ischemic adult cardiac and skeletal muscle results from the processes of angiogenesis, arteriogenesis and vasculogenesis. Therapeutic angiogenesis describes an emerging field of cardiovascular medicine whereby new blood vessels are induced to grow to supply oxygen and nutrients to cardiac or skeletal muscle rendered ischemic as a result of progressive atherosclerosis. Various techniques have been utilized to promote new blood vessel growth in the heart and extremities, including mechanical means such as surgical or percutaneous myocardial laser revascularization, angiogenic growth factor therapies involving members of the vascular endothelial growth factor and fibroblast growth factor families, and more recently, cellular-based therapies using stem cells known as endothelial progenitor cells or angioblasts. The following review discusses each of these treatment strategies in detail including both preclinical and clinical data for their use in peripheral arterial and coronary artery disease.     

Angiogenic therapy for coronary artery and peripheral arterial disease

Neovascularization in chronically ischemic adult cardiac and skeletal muscle results from the processes of angiogenesis, arteriogenesis and vasculogenesis. Therapeutic angiogenesis describes an emerging field of cardiovascular medicine whereby new blood vessels are induced to grow to supply oxygen and nutrients to cardiac or skeletal muscle rendered ischemic as a result of progressive atherosclerosis. Various techniques have been utilized to promote new blood vessel growth in the heart and extremities, including mechanical means such as surgical or percutaneous myocardial laser revascularization, angiogenic growth factor therapies involving members of the vascular endothelial growth factor and fibroblast growth factor families, and more recently, cellular-based therapies using stem cells known as endothelial progenitor cells or angioblasts. The following review discusses each of these treatment strategies in detail including both preclinical and clinical data for their use in peripheral arterial and coronary artery disease.     

Decreased angiogenesis and arthritic disease in rabbits treated with an αvβ3 antagonist

Rheumatoid arthritis (RA) is an inflammatory disease associated with intense angiogenesis and vascular expression of integrin αvβ3. Intra-articular administration of a cyclic peptide antagonist of integrin αvβ3 to rabbits with antigen-induced arthritis early in disease resulted in inhibition of synovial angiogenesis and reduced synovial cell infiltrate, pannus formation, and cartilage erosions. These effects were not associated with lymphopenia or impairment of leukocyte function. Furthermore, when administered in chronic, preexisting disease, the αvβ3 antagonist effectively diminished arthritis severity and was associated with a quantitative increase in apoptosis of the angiogenic blood vessels. Therefore, angiogenesis appears to be a central factor in the initiation and persistence of arthritic disease, and antagonists of integrin αvβ3 may represent a novel therapeutic strategy for RA.     

Effective treatment of vascular endothelial growth factor refractory hindlimb ischemia by a mutant endothelial nitric oxide synthase gene

Gene delivery of angiogenic growth factors is a promising approach for the treatment of ischemic cardiovascular diseases. However, success of this new therapeutic principle is hindered by the lack of critical understanding as to how disease pathology affects the efficiency of gene delivery and/or the downstream signaling pathways of angiogenesis. Critical limb ischemia occurs in patients with advanced atherosclerosis often exhibiting deficiency in endothelial nitric oxide production. Similar to these patients, segmental femoral artery resection progresses into severe ischemic necrosis in mice deficient in endothelial nitric oxide synthase (ecNOS-KO) as well as in balb/c mice. We used these models to evaluate the influence of severe ischemia on transfection efficiency and duration of transgene expression in the skeletal muscle following plasmid injection in combination with electroporation. Subsequently, we also explored the potential therapeutic effect of the phosphomimetic mutant of ecNOS gene (NOS1177D) using optimized delivery parameters, and found significant benefit both in ecNOS-KO and balb/c mice. Our results indicate that NOS1177D gene delivery to the ischemic skeletal muscle can be efficient to reverse critical limb ischemia in pathological settings, which are refractory to treatments with a single growth factor, such as vascular endothelial growth factor.     

The inhibition of vascular smooth muscle cell migration by peptide and antibody antagonists of the alphavbeta3 integrin complex is reversed by activated calcium/calmodulin- dependent protein kinase II.

The migration of vascular smooth muscle cells (VSMCs) is thought to play a key role in the pathogenesis of many vascular diseases and is regulated by soluble growth factors/ chemoattractants as well as interactions with the extracellular matrix. We have studied the effects of antibodies to rat beta3 and human alphavbeta3 integrins on the migration of VSMCs. Both integrin antibodies as well as cyclic RGD peptides that bind to the vitronectin receptors alphavbeta3 and alphavbeta5 significantly inhibited PDGF-directed migration. This resulted in a reduction in the accumulation of inositol (1,4,5) trisphosphate and the activation of calcium/calmodulin-dependent protein kinase II (CamKII), an important regulatory event in VSMC migration identified previously. PDGF-directed VSMC migration in the presence of the anti-integrin antibodies and cyclic RGD peptides was restored when intracellular CamKII activity was elevated by either raising intracellular calcium levels with the ionophore, ionomycin, or infecting with a replication-defective recombinant adenovirus expressing a constitutively activated CamKII cDNA (AdCMV.CKIID3). Rescue of rat VSMCs was also observed in stably transfected cell lines expressing constitutively activated but not wild-type CamKII. These observations identify a key intermediate in the regulation of VSMC migration by outside-in signaling from the integrin alphavbeta3.     

Adenovirus-mediated E2-EPF UCP Gene Transfer Prevents Autoamputation in a Mouse Model of Hindlimb Ischemia

E2-EPF ubiquitin carrier protein (UCP) stabilizes hypoxia-inducible factor-1α (HIF-1α) inducing ischemic vascular responses. Here, we investigated the effect of UCP gene transfer on therapeutic angiogenesis. Adenovirus-encoded UCP (Ad-F-UCP) increased the expression of vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2) in cells and mice. Conditioned media from UCP-overexpressing cells promoted proliferation, tubule formation, and invasion of human umbilical-vascular-endothelial cells (HUVECs), and vascularization in chorioallantoic membrane (CAM) assay. Ad-F-UCP increased the vessel density in the Martigel plug assay, and generated copious vessel-like structures in the explanted muscle. The UCP effect on angiogenesis was dependent on VEGF and FGF-2. In mouse hindlimb ischemia model (N = 30/group), autoamputation (limb loss) occurred in 87% and 68% of the mice with saline and Ad encoding β-galactosidase (Ad-LacZ), respectively, whereas only 23% of the mice injected with Ad-F-UCP showed autoamputation after 21 days of treatment. Ad-F-UCP increased protein levels of HIF-1α, platelet-endothelial cell adhesion molecule-1 (PECAM-1), smooth muscle cell actin (SMA) in the ischemic muscle, and augmented blood vessels doubly positive for PECAM-1 and SMA. Consequently, UCP gene transfer prevented muscle degeneration and autoamputation of ischemic limb. The results suggest that E2-EPF UCP may be a target for therapeutic angiogenesis.     

A growth factor mixture that significantly enhances angiogenesis in vivo

Studies of therapeutic angiogenesis have generally focused on single growth factor strategies. However, multiple factors participate in angiogenesis. We evaluated the angiogenic potential of a growth factor mixture (GFm) derived from bovine bone. The major components of GFm (SDS-polyacrylamide gel electrophoresis, mass spectrometry, and Western blot) include transforming growth factor-beta1-3, bone morphogenic protein-2-7, and fibroblast growth factor-1. GFm was first shown to induce an angiogenic response in chorioallantoic membranes. Next, myocardial ischemia was induced in 21 dogs (ameroid) that were randomized 3 weeks later to received GFm 1 mg/ml (I), GFm 10 mg/ml (II), or placebo (P) (with investigators blinded to conditions) injected in and adjacent to ischemic myocardium. Dogs were assessed 6 weeks later using quantitative and semiquantitative measures. There were GFm concentration-dependent improvements in distal left anterior descending artery (LAD) opacification by angiography (P: 0.4 +/- 0.2, I: 1.1 +/- 0.14, II: 1.6 +/- 0.3, angiographic score p = 0.014). Histologically, there was also concentration-dependent vascular growth response of relatively large vessels (P: 0.21 +/- 0.15, I: 1.00 +/- 0.22, II: 1.71 +/- 0.18, vascular growth score p = 0.001). Resting myocardial blood flow (colored microspheres) was not significantly impaired in any group. However, maximum blood flow (adenosine) was reduced in ischemic territories and did not improve in GFm-treated hearts. GFm, a multiple growth factor mixture, is a potent angiogenic agent that stimulates large vessel growth. Although blood flow did not improve during maximal vasodilatory stress, large intramyocardial collateral vessels developed and angiographic visualization of the occluded distal LAD improved significantly. The use of multiple growth factors may be an effective strategy for therapeutic angiogenesis provided a more effective delivery strategy is devised that can achieve improved maximum blood flow potential.